Acinetobacter Calcoaceticus Strain Research Articles

Partial purification and characterization of a non-cyanobacterial cyanophycin synthetase from Acinetobacter calcoaceticus strain ADP1 with regard to substrate specificity, substrate affinity and binding to cyanophycin.

This study reports, for the first time, purification and biochemical characterization of a cyanophycin synthetase from a non-cyanobacterial strain. Cyanophycin synthetase of Acinetobacter calcoaceticus strain ADP1 was purified 69-fold from recombinant Escherichia coli by two chromatographic steps and one novel affinity step utilizing the Mg(2+)-dependent binding of the enzyme to cyanophycin. Unlike cyanobacterial cyanophycin synthetases characterized so far, the purified enzyme from A. calcoaceticus strain ADP1 did not accept lysine as an alternative substrate to arginine. The apparent K(m)-values for arginine (47 microM) and aspartic acid (240 microM) were similar to those of known cyanophycin synthetases from cyanobacteria, but this enzyme had a slightly higher affinity for aspartic acid. In addition, the two different ATP-binding sites of the enzyme were characterized independently of each other with respect to K(m) values for ATP. The ATP-binding site responsible for the addition of arginine was found to have a much higher affinity for ATP (38 microM) than that responsible for the addition of aspartate (210 mM). Furthermore, the binding of the enzyme to the two possible forms of cyanophycin granule polypeptide (CGP), CGP-Asp and CGP-Arg, was studied. While both forms bound around 30-40 % of the enzyme activity present under the assay conditions, binding was Mg(2+)-dependent in the case of CGP-Asp. Two-dimensional gel electrophoresis revealed that both forms of cyanophycin were equally abundant in cyanophycin-accumulating cells of A. calcoaceticus ADP1.

Microbial hydroxylation of indole to 7-hydroxyindole by Acinetobacter calcoaceticus strain 4-1-5.

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.

Prevalence and in-vitro antimicrobial susceptibility patterns of Acinetobacter strains isolated from patients in intensive care units.

Fifty-six Acinetobacter species strains (49 Acinetobacter baumanii, 5 Acinetobacter calcoaceticus, 2 Acinetobacter iwoffii) were detected using both conventional methods and gas chromatography of bacterial fatty acids with the MIDI Sherlock Microbial Identification System. The susceptibilities of these strains to 16 antimicrobial agents were investigated by the disc-diffusion method according to the National Committee for Clinical Laboratory Standards. The production of extended-spectrum beta-lactamases (ESBLs) and inducible beta-lactamases (IBLs) by the strains were investigated by the double-disc-synergy and disc-approximation methods, respectively. Imipenem was the most effective agent for Acinetobacter baumanii strains (95.9% of strains were sensitive), while meropenem and netilmicin showed moderate activity (87.7% and 79.6% of strains, respectively, responded). Acinetobacter baumanii strains were less sensitive to cefoperazone-sulbactam (53.1%), ofloxacin (51.0%), ciprofloxacin (42.8%), and amikacin (36.7%). Acinetobacter calcoaceticus and Acinetobacter iwoffii strains were sensitive to imipenem, meropenem and netilmicin. IBLs and ESBLs were produced, respectively, by 8.9% and 7.1% of all bacterial strains. The strains isolated were sufficiently sensitive to imipenem, but not to ofloxacin or ciprofloxacin, and were very resistant to amikacin.

Purification and Characterization of Membrane-bound Quinoprotein Quinate Dehydrogenase

Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant holo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3. This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 nm. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6-7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate.

Bioengineered emulsans from Acinetobacter calcoaceticusRAG-1 transposon mutants.

Transposon mutants of Acinetobacter calcoaceticus strain RAG-1 were studied in an effort to control fatty acid (FA) substitution patterns of emulsan, a bioemulsifier secreted by the organism. The disrupted genes, involved in the biosynthetic pathways of biotin, histidine, cysteine or purines, influenced the level and types of FAs incorporated into emulsan. The structural variants of emulsan generated by the transposon mutants were characterized for yield, FA content, molecular weight, and emulsification behavior when grown on a series of FAs of different chain lengths from C11 to C18. Yields of emulsan from the transposon mutants were found to be lower than the parent strain and depended on the type of FA used to supplement the growth medium. Mutants 13D (His-) and 52D (Cys-) grown on LB plus C16 or C14, respectively, exhibited enhanced emulsifying activity compared to A. calcoaceticus RAG-1. The presence and composition of long chain FAs on the polysaccharide backbone influenced emulsification behavior: particularly a high mole percentage of C16 (48%) and C18 (42%). The results provide important insight into the bioengineering of bioemulsifier-producing microorganisms and provide a path towards highly tailored novel amphipathic structures to utilize as biodegradable in environmental, biomedical, and personal care applications.

Degradation of Mineral Oil with a Strain of Acinetobacter calcoaceticus

The Acinetobacter calcoaceticusstrain TM-31 has been isolated from a microbial assemblage of a pilot plant that purifies waste water polluted with mineral oil. This strain is capable of efficient degradation of components of mineral oil (alkanes, isoalkanes, and alkyl residues of the naphthene and arene fraction). The strain bears stably inherited plasmids of sizes 120, 9, and 8 kb, which can be transferred into plasmid-free cells of the parental strain and into bacteria of the genusPseudomonasand ensure the degradation of hexadecane and mineral oil.

Engineering of Heterologous Cytochrome P450 in Acinetobacter sp.: Application for Pollutant Degradation

Many organisms do not contain the necessary biochemical armoury to carry out the initial oxidative attack of many pollutant chemicals. In the present study, Acinetobacter sp. strain BD413 has been genetically engineered to express the cytochrome P450 xenobiotic-metabolising enzyme CYP105D1 from Streptomyces griseus that has in its repertoire a diverse array of organic pollutants. Further, it is shown that the transformed Acinetobacter calcoaceticus strain BD413 can grow on pollutants unlike control bacteria not expressing cytochrome P450 and that was reflected in release of radiolabel with growth on radiolabelled chlortoluron. We show that cytochrome P450 can enhance the biodegrading repertoire of A. calcoaceticus and discuss the application of such results to bioremediation strategies.

A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds.

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 microgram/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed.

Antibiotic resistance among gram-negative nosocomial pathogens in the intensive care unit: results of 6-year body-site monitoring

Results of 6-year body-site monitoring in an intensive care unit (ICU) are presented and antimicrobial resistance of gram-negative isolates analyzed. The study included 622 patients. Six hundred thirty-five bacterial isolates—causes of nosocomial sepsis, pneumonia, and urinary tract infections (UTIs)—were tested during the study. Gram-negative bacteria were the predominant isolates, causing 65% of cases of sepsis, 78.7% of pneumonias, and 70.2% of UTIs. Gram-negative isolates (454) were highly resistant to antimicrobials commonly used in the ICU, with the exception of imipenem. Resistance was 1.1% among pathogens responsible for UTIs, 6.7% among those causing sepsis, and 13.6% among those responsible for pneumonia. Klebsiella pneumoniae associated with pneumonia and sepsis was significantly less resistant to ciprofloxacin than were isolates from urine (22.8% and 13.9%, respectively, vs 44.4%). Pseudomonas aeruginosa strains responsible for pneumonia were less resistant to ceftadizime than were isolates causing sepsis and UTI (35.7% vs 51.3% and 51.5%, respectively). Acinetobacter calcoaceticus strains associated with UTI were significantly more resistant to netilmicin than were strains responsible for sepsis and pneumonia (83.3% vs 40.3% and 42.6%, respectively). The study confirmed that in addition to focused microbiologic surveillance, multiple-body-site monitoring can provide unique information about the sensitivity of the pathogens involved. The results suggest that antimicrobial resistance among nosocomial pathogens depends on the site of infection or the type of microbiologic specimen.

Structural and serological characterisation of the O-specific polysaccharide from lipopolysaccharide of Acinetobacter calcoaceticus strain 7 (DNA group 1).

S-form lipopolysaccharide was isolated by phenol/water extraction from a strain of Acinetobacter calcoaceticus (DNA group 1 ). The structure of the O-antigenic polysaccharide was determined by compositional analysis and NMR spectroscopy of the de-O-acylated lipopolysaccharide. The isolated polysaccharide obtained after hydrolysis of lipopolysaccharide in 0.01 M trifluoroacetic acid has the following structure: [STRUCTURE IN TEXT] in which Pyr is pyruvate. The O-acetyl substitution of D-Gal was non-stoichiometric. The O-antigen was specifically recognised in western blots by polyclonal rabbit antisera.

Natural transformation in river epilithon.

Natural transformation was demonstrated in unenclosed experiments incubated in river epilithon. Strains of Acinetobacter calcoaceticus were transformed to prototrophy by either free DNA (lysates) or live donor cells. The sources of transforming DNA and recipient culture were immobilized on filters, secured to stones, and incubated midstream in the river. The transfer frequency generally increased with temperature. No transfer was detected in the river Taff below 10 degrees C. The age of the recipient culture affected the transformation frequencies in situ but did not significantly affect the transfer frequency on laboratory media. Transformation of recipient cultures which had been incorporated into the natural epilithic biofilm and transformation of the plasmid pQM17 in situ were also demonstrated. This study provides the first direct evidence of natural transformation in situ of bacteria incorporated into an indigenous community.

Effect of nitrogen sources on the regulation of extracellular lipase production in Acinetobacter calcoaceticus strains

Extracellular lipase production and lipA expression were studied in A.calcoaceticus strains in relation to the nitrogen sources. Addition of casamino-acids or tryptone improved 2–3 times lipase yield as compared to ammonium, yeast extract or proteose-peptone. Further increase in lipase yield and enzyme stability were achieved when ammonium was also added. Differences in yields were not related to lipA transcription. Proteolytic activity was detected in culture broths simultaneous with lipase degradation. Post- transcriptional processes including enzyme protection, inactivation and secretion must be considered as important factors affecting lipase production.

Emulsifier production and microscopical study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcoaceticus MM5

A bacterial strain was isolated from a sample of contaminated heating oil and identified as a strain of Acinetobacter calcoaceticus, named MM5. The bacterial isolate was able to grow on petroleum derivatives and brought about an emulsification of those compounds. A bioemulsifier was extracted from the culture medium of MM5 strain and partially characterized. This compound was able to emulsify petroleum fuels and both aliphatic and aromatic pure hydrocarbons and was stable over a wide range of temperatures. Studies developed by light, scanning electron and transmission electron microscopy showed that, during the growth on petroleum derivatives, the microorganisms were orientated on the surface of drops enclosed in a skin or membranous polymer produced by the bacteria. These droplets may represent the hydrocarbon/water emulsion of the liquid culture. The growth of A. calcoaceticus MM5 on media containing both hydrocarbon and water-soluble substrates as carbon sources also results in the formation of a film, consisting of amorphous and membranous layers. The bacteria were connected to the biofilm and showed intercellular contacts through cell-surface appendages, forming a complex network. The importance of the biofilms for bacterial adhesion to oil droplets and for its nourishment is discussed.

Cloning of catBCIJFD genes for catechol degradation into chromosomal pobA and genetic stability of the recombinant Acinetobacter calcoaceticus.

A possible obstacle in the development of hybrid strains of Acinetobacter calcoaceticus by the introduction of a metabolic pathway into the chromosome is genetic instability of the resulting recombinant strains. Therefore, the possibility that the pobA gene can be used as a chromosomal cloning site where the transposed genes can be maintained and expressed, was explored in this study. For this purpose, two model hybrid strains of A. calcoaceticus were created, in which a DNA fragment carrying catBCIJFD genes for catabolic degradation of catechol was inserted into pobA in opposite directions of each other, and their genetic stabilities were experimentally examined. Our data demonstrated that the stability of the genes neighboring the insertions depends on the orientations of the insertions. Also, the data further indicated that the functional metabolic pathways introduced into pobA can be expressed successfully as far as the insertion is engineered in an appropriate way. Concurrently, it was proposed that the pobA can be used as a chromosomal cloning site, and that introduction of an useful metabolic pathway into pobA may offer considerable promise to the construction of a hybrid strain with improved metabolic capabilities.

Degradation of trans-ferulic and p-coumaric acid by Acinetobacter calcoaceticus DSM 586

Cell suspensions of Acinetobacter calcoaceticus strain DSM 586 and DSM 590 were able to grow on benzoic, p-hydroxybenzoic and vanillic acid as sole carbon source. Testing the utilization of trans-ferulic and p-coumaric acid, we found that the sole A. calcoaceticus DSM 586 efficiently degraded the lignocellulose related monomers. Cells induced with trans-ferulic acid were able to oxidize trans-ferulic, p-coumaric, vanillic, p-hydroxybenzoic and protocatechuic acid at rates higher than the uninduced culture. The same activity was found in the p-coumaric acid induced culture. Two aromatic compounds, vanillic and p-hydroxybenzoic acid, were isolated from culture filtrates of trans-ferulic and p-coumaric acid grown cells, respectively, and further characterized by high performance liquid chromatography, 1H- and 13C-nuclear magnetic resonance and ultraviolet spectrophotometry. Cell extracts of trans-ferulic or p-coumaric acid induced cultures were shown to rapidly convert protocatechuic acid to β-car☐ymuconic acid. Moreover, A. calcoaceticus DSM 586 produced high levels of protocatechuic 3,4-dioxygenase compared to cathecol 1,2-dioxygenase and gentisate 1,2-dioxygenase in the degradation of trans-ferulic or p-coumaric acid. Based upon these results, a reaction sequence for the complete degradation of trans-ferulic and p-coumaric acid in A. calcoaceticus DSM 586 is proposed.

Spontaneous mutations in pcaH and -G, structural genes for protocatechuate 3,4-dioxygenase in Acinetobacter calcoaceticus

Bacteria containing spontaneous null mutations in pcaH and -G, structural genes for protocatechuate 3,4-dioxygenase, were selected by exposure of an Acinetobacter calcoaceticus strain to physiological conditions in which expression of the genes prevents growth. The parental bacterial strain exhibits high competence for natural transformation, and this procedure was used to characterize 94 independently isolated spontaneous mutations. Four of the mutations were caused by integration of a newly identified insertion sequence, IS1236. Many (22 of 94) of the mutations were lengthy deletions, the largest of which appeared to eliminate at least 17 kb of DNA containing most of the pca-qui-pob supraoperonic gene cluster. DNA sequence determination revealed that the endpoints of four smaller deletions (74 to 440 bp in length) contained DNA sequence repetitions aligned imprecisely with the sites of mutation. Analysis of direct and inverted DNA sequence repetitions associated with the sites of mutation suggested the existence of DNA slippage structures that make unhybridized nucleotides particularly susceptible to mutation.

Evolutionary divergence of pobA, the structural gene encoding p-hydroxybenzoate hydroxylase in an Acinetobacter calcoaceticus strain well-suited for genetic analysis

The pobA gene encoding p-hydroxybenzoate hydroxylase (PobA) from Acinetobacter calcoaceticus has been developed as a genetic tool for the analysis of structure-function relationships in this enzyme. By exploiting the favorable genetic system of A. calcoaceticus strain ADP1, it is possible both to select and to map mutations which disturb PobA activity; characterization and sequence determination of mutants derived in this manner may complement site-directed studies with the homologous Pseudomonas aeruginosa gene. We have determined the nucleotide (nt) sequence of A. calcoaceticus pobA and performed a systematic comparison of the deduced amino acid (aa) sequence with that of the PobA enzyme from Pseudomonas fluorescens, for which the three-dimensional structure is known. Despite a 26% difference in the G + C content of the homologous genes, constraints against structural divergence of the proteins were revealed by an overall identity of 62.4% in the aligned aa sequences of PobA. Clusters of identical sequence occur at previously identified sites of ligand binding and at regions associated with subunit-subunit interaction. Based on the conservation of specific residues involved in flavin binding, we have assembled a consensus sequence for nicotinamide-flavoprotein monooxygenases which differs from that of the oxidoreductase class of flavoproteins. In addition to the conserved regions shared by the two PobA homologs, there are isolated pockets of divergence. The nt sequence divergence in one such region within the A. calcoaceticus gene can be attributed to the acquisition of short nt sequence repetitions.

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N

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 Würster'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.

Epidemiological role of arthropods detectable in health facilities

A total of 161 arthropod specimens were collected from 55 sites in a health care facility during July and September 1990. Of the 116 bacterial isolates obtained from their body surfaces 6% were from parasites (mosquitoes), 59% from eusynanthropic arthropods (Tenebrionid beetles, flies, German cockroaches, wasps), 16% from hemisynanthropic arthropods (ants, spiders) and 19% from occasionally encountered insects (non-biting midges, moths, beetles). Most (88%) of the isolated bacteria were Gram-negative rods of the species E. coli, Enterobacter, Klebsiella, Citrobacter, Proteus, Serratia, Pseudomonas and Acinetobacter. Gram-positive cocci accounted for 13% of isolates and were primarily represented by coagulase-negative staphylococci. The highest isolation rates were from body surfaces of flies, German cockroaches, non-biting midges (Chironomids) and Tenebrionid beetles. About one third of all isolates were resistant to more than three antimicrobials using a standard disc diffusion assay. The presence of multiple resistance to antibiotics was observed in two thirds of Enterobacter isolates, namely those of Enterobacter cloacae from the body surface of German cockroaches, in 13% of Citrobacter spp and in 8% of Klebsiella spp as well as Acinetobacter calcoaceticus strains. Strains of Morganella and Hafnia species were very infrequent but all of them shared resistance to the antibiotics tested. In contrast, strains of Serratia spp were relatively antibiotic-sensitive. The group of isolated Gram-positive organisms was represented by two strains of Staphylococcus hominis and one strain of Enterococcus sp, all of them were multiply-resistant to antimicrobials.

Mechanisms of resistance to β-lactam antibiotics in Acinetobacter calcoaceticus

Examination of 12 strains of Acinetobacter calcoaceticus revealed that the strains expressed different constitutive levels of beta-lactamase. Mutants resistant to cefoxitin, cefoperazone or ceftazidime were selected from a strain producing a low level of beta-lactamase. All the mutants showed no change in expression of beta-lactamase, but produced penicillin-binding proteins with altered expression and/or affinity for beta-lactams. In addition, the outer membrane of the mutants showed decreased permeability (40-80% that of the parent strain) towards small hydrophilic solutes, together with diminished production of a 46.5 kDa porin protein. It was concluded that the enhanced resistance to beta-lactams in the A. calcoaceticus mutants was the result of interplay between the altered penicillin binding proteins and the reduced outer membrane permeability.