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In Acinetobacter calcoaceticus, a Gram-negative bacterial species, a soluble insulin-degrading proteinase, located in the periplasm as well as in the cytosol, could be established. The periplasmic and cytosolic enzymes agree in their inhibition pattern, pH-optimum and molecular weight. The insulin-degrading enzyme of Acinetobacter calcoaceticus resembles the corresponding proteinases of Escherichia coli. It is a metalloproteinase with a pH-optimum in the neutral range and can be reactivated by divalent ions after EDTA-inhibition, but it is not entirely identical with any of the described proteinases of Escherichia coli in its inhibitory behaviour.
Three Acinetobacter calcoaceticus transformation-deficient mutants, obtained by insertional mutagenesis with the nptll gene, have been characterized physiologically. One mutant (AAC211) was found to be completely transformation deficient, while two others, AAC213 and AAC214, were severely impaired in transformation efficiency (100-1000 times lower than the wild type). The latter applied to both chromosomal as well as plasmid DNA. Analysis of the chromosomal DNA fragments flanking the nptll gene in the mutants showed that mutants AAC213 and AAC214 had an insertion of the nptll gene in the same chromosomal region, but that they were the result of two independent mutational events, whereas the insertion in mutant AAC211 was at a different position. None of the three mutants showed phenotypic or genotypic characteristics typical of a RecA-deficient strain.
Acinetobacter calcoaceticus BD413 produces variable amounts of an exocellular lipase that becomes rapidly inactivated upon secretion. To achieve high yield and protect the enzyme, we assayed the addition of several inert compounds to cell-free supernatants, cell fractions, and whole cultures. Glass beads, poly(ethylene glycol) 600, Triton X-100, saccharose, gum arabic, and beta-cyclodextrin were among the compounds tested. beta-Cyclodextrin and gum arabic (and saccharose to a lesser extent) were effective enzyme stabilizers in cell-free supernatants, while gum arabic, glass beads, and Triton X-100 improved lipase secretion from cells, and, therefore, total lipase yield (30-50%, according to the additive). In whole cultures, beta-cyclodextrin was the most effective additive, particularly in combination with glass beads or gum arabic. Indeed, cultures containing beta-cyclodextrin plus gum arabic were able to maintain 95% (+/- 1.5%) of the initial lipase activity for more than 16 h, while control cultures with no additives maintained only 10% (+/- 4%) of the enzyme activity after the same period. In conclusion, the addition of inert compounds in cultures may be considered a useful approach for achieving increased yield and lipase stabilization, amenable for downstream processing.
In Acinetobacter calcoaceticus the seven genes coding for the enzymes responsible for tryptophan synthesis map at three chromosomal locations. Two three-gene clusters, one (trpGDC) specifying the small subunit of anthranilate synthase, phosphoribosyl transferase, and indoleglycerol phosphate synthase and the other (trpFBA) specifying phosphoribosyl anthranilate isomerase and both tryptophan synthase subunits, are not linked to each other or to the trpE gene specifying the large anthranilate synthase subunit. When regulation of trp gene expression is studied in the wild type, only the level of the trpF gene product decreases upon addition of tryptophan to the medium. Tryptophan starvation of tryptophan auxotrophs, however, results in increased levels of all the tryptophan enzymes; this and additional evidence suggests that the expression of all the trp genes is subject to repression. The trpGDC genes are coordinately controlled, and the trpE gene is regulated in parallel with them. The trpFBA genes are controlled neither coordinately nor in parallel with the other trp genes, but respond proportionally when compared with each other. So far, two types of constitutive mutants have been found. The first class of mutants apparently occurs in the structural gene for a repressor protein; this repressor locus is unlinked to any of the biosynthetic trp genes and affects only the expression of trpE and the trpGDC cluster. The second class contains mutants closely linked to the trpGDC region; they overproduce only the gene products of this cluster.
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.
Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.
Acinetobacter calcoaceticus MTC 127 was able to grow on catechin and protocatechuic acid (PCA) as sole carbon source. Cells induced with catechin oxidized catechin and PCA at rates higher than cells of uninduced cultures. Two aromatic compounds, PCA and phloroglucinol carboxylic acid (PGCA) were isolated from culture filtrate of cells grown in catechin and characterized by infrared spectrometry and high performance thin-layer chromatography. Moreover, A. calcoaceticus MTC 127 produced high levels of PCA compared to PGCA in the degradation of catechin. Based upon these results, a pathway for the degradation of (+)-catechin in A. calcoaceticus MTC 127 is proposed. Enzymes extracted from catechin-induced culture showed catechin oxygenase (cox) and protocatechuate 3,4-dioxygenase (pcd) activities. Catechin oxygenase was purified by column chromatography and SDS-PAGE analysis showed a single band with an apparent molecular weight of 47 kDa.
The Acinetobacter calcoaceticus strain TM-31 has been isolated from a microbial assemblage of a pilot plant purifying 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 genus Pseudomonas and ensure the degradation of hexadecane and mineral oil.
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 108,000 (gel filtration) or 112,000 (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).
Acinetobacter calcoaceticus BD413 was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron-scavenging phenotype was associated with the secretion of iron-repressible catechol and the induction of a group of six outer membrane proteins with molecular weights ranging from 34 to 85 kDa. The amount of catechol produced was dependent on medium composition and iron stringency. The relation between iron limitation and lipase production was studied at the level of lipA transcription and extracellular lipase activity. In minimal medium, iron limitation slightly affected lipA expression but decreased exo-lipase activity significantly. However, if iron limitation and rich nitrogen sources were simultaneously present in the culture media, the production of lipase was increased approximately 4 times.
Acinetobacter calcoaceticus NCIB 8250 utilizes phenol as sole source of carbon and energy via an ortho-cleavage pathway. The presence of ethanol in mixed substrate cultivations repressed the utilization of phenol. In fed batch cultivation the phenol tolerance was increased at least 2-fold. Maximum degradation rates of 150 mg phenol/(1 h) and 280 mg phenol/(g h), respectively were observed. Phenol hydroxylase is induced by its substrate and in parallel the catechol-1,2-dioxygenase is detectable. The presence of active phenol hydroxylase is strongly connected with the phenol degradation. Using a spectrophotometric enzyme assay the partially purified phenol hydroxylase was characterized with respect to kinetic parameters. The apparent Km values for phenol, FAD and NADPH were estimated to be 147 microM, 35 microM and 416 microM, respectively. Both FAD and NADPH were essential for maximum activity of the cytoplasmically localized enzyme. No substrate inhibition of phenol hydroxylase by phenol was observed up to 0.8 mM. The pH and temperature optima were pH 7.8 and 33 degrees C, respectively. The partially purified enzyme showed a broad substrate specificity. It hydroxylated the three isomeric cresols, chlorophenols and methylated chlorophenols. Pyrogallol, 3,4-dihydroxy-L-phenylalanine and resorcinol were oxygenated with higher rates than phenol. With the exception of phenol all other enzyme substrates tested did not serve as growth substrates.
Cells of Acinetobacter calcoaceticus 69-V, a species able to metabolize a range of aliphatic hydrocarbons and alcohols, were confronted with ethanol, butanol, hexanol or heat shock during growth on acetate as sole source of carbon and energy. The primary alcohols and the heat shock led to the synthesis of new proteins or amplified expression of specific, common and general proteins, which were detected by silver staining after two-dimensional gel electrophoresis. Some of the alcohol-inducible proteins were identified as heat shock proteins by comparing protein patterns of alcohol-shocked cells with those of heat-shocked cells, and by N-terminal amino acid sequencing. DnaK was found to be amplified after all treatments, but GroEI only after heat shock and ethanol treatment. The N-terminal amino acid sequence of the protein, which was considerably amplified after alcohol treatment and heat shock, shows homology to HtpG (high temperature protein G). Some of the heat shock proteins induced by ethanol differ from those induced by butanol and hexanol, suggesting there are at least two different signals for the induction of some heat shock proteins by primary alcohols. This could be due to the different localization of ethanol, butanol and hexanol in the membrane, or because higher cytoplasmic concentrations of ethanol than of butanol or hexanol were applied in these tests in order to keep concentrations of the alcohols in the membrane roughly similar. Besides heat shock proteins, a group of proteins were observed which were only induced by butanol and hexanol, possibly indicating the existence of a further defense mechanism against high concentrations of hydrophobic substrates preventing protein denaturation and membrane damage.
Acinetobacter spp. are uncommon etiologic agents of prosthetic valve endocarditis. Two patients with Acinetobacter calcoaceticus subsp. lwoffi prosthetic valve endocarditis are described. The patients were successfully treated with antibiotics (cefotaxime sodium and gentamicin sulfate); thus, we suggest medical treatment rather than early valve replacement in this particular type of infection.
Mutants of Acinetobacter calcoaceticus LMD79.41 were isolated that are defective in the synthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ). A gene bank of the wild-type. A. calcoaceticus genome was constructed with the binary plasmid system pLV21-RP4 delta Km. The DNA of A. calcoaceticus LMD79.41 was partially digested with Sau3A, and fragments of about 15 kilobases were inserted into the BamHI site of pLV21. The hybrid plasmids maintained in Escherichia coli were transferred by conjugation to the PQQ- mutants of A. calcoaceticus. One hybrid plasmid was isolated that complements all isolated PQQ- mutants. Subcloning of this plasmid in the vector pRK290 resulted in an insert of 5 kilobases on which at least four different genes involved in PQQ synthesis could be indicated. With Tn5 insertions the four PQQ genes were mapped, and it was shown that these genes are most probably located in three operons.
Serotyping of Acinetobacter calcoaceticus by direct immunofluorescence and a capsule swelling reaction is described. One hundred isolates, including 12 from an outbreak in a neonatal department, were studied. Ninety five per cent of the isolates were typable by immunofluorescence and could be divided into 30 separate types, but 42.1% of typable strains, including 11 from the outbreak, were of one type. Typing results by the capsular swelling reaction generally followed those of immunofluorescence methods.
Mutants of Acinetobacter calcoaceticus EBF 65/65 that could not grow on intermediates of the mandelate or benzyl alcohol pathways were isolated and in some cases the enzymic lesions were identified. Several catabolic markers were mapped using the plasmid pAV1. The mandelate genes appeared to be clustered near the auxotrophic marker phe-1 but were not all contiguous with each other. The gene responsible for the appearance of the novel L(+)-mandelate dehydrogenase appeared to be close to a gene responsible for the activity of the original D(-)-mandelate dehydrogenase.
Cells of Acinetobacter calcoaceticus strain 69-V contain an aminopeptidase that cleaves L-leucine amide, leucylglycine or leucine hydrazide with high efficiency. Leucine 4-nitroanilide and hydrazide are hydrolyzed to less than 0.1% and 1%, resp. of leucine amide. Grown on acetate-NH4+ medium the activity of the enzyme in the cytoplasm is increased 5-fold compared with cells grown on a casamino acid medium or on yeast extract. In these cases the specific activity of the unpurified enzyme is about 5 nkat/mg for the cytoplasmic and membrane-bound enzyme species as well. Up to 30% of the aminopeptidase activity were found mainly in intracytoplasmic membranes, less in cytoplasmic membranes and only traces in outer membranes, presumably as contaminations. It is solubilized by detergents but not by high salt concentrations. An addition of antipain or Z-Ala2-Phe-CH3 before cell rupture did not change the distribution of the enzyme. A mixture of EDTA and 1.10-phenanthroline diminished the membrane-bound enzyme from 11.4% to 4.3% and leupeptin or E-64 increased it to 20%. The enzyme is regarded as leucine aminopeptidase (LAP) bound to intracytoplasmic membranes.