Calcoaceticus NCIB8250 Research Articles

Structure Guided Design of Protein Biosensors for Phenolic Pollutants.

Phenolic aromatic compounds are a major source of environmental pollution. Currently there are no in situ methods for specifically and selectively detecting these pollutants. Here, we exploit the nature's biosensory machinery by employing Acinetobacter calcoaceticus NCIB8250 protein, MopR, as a model system to develop biosensors for selective detection of a spectrum of these pollutants. The X-ray structure of the sensor domain of MopR was used as a scaffold for logic-based tunable biosensor design. By employing a combination of in silico structure guided approaches, mutagenesis and isothermal calorimetric studies, we were able to generate biosensor templates, that can selectively and specifically sense harmful compounds like chlorophenols, cresols, catechol, and xylenols. Furthermore, the ability of native protein to selectively sense phenol as the primary ligand was also enhanced. Overall, this methodology can be extended as a suitable framework for development of a series of exclusive biosensors for accurate and selective detection of aromatic pollutants from real time environmental samples.

Improvement of an E. coli bioreporter for monitoring trace amounts of phenol by deletion of the inducible σ54-dependent promoter

An Escherichia coli bioreporter harboring the phenol-inducible mphK promoter (P(mphK)) from Acinetobacter calcoaceticus PHEA-2 fused to a beta-galactosidase gene (lacZ) and the regulator gene (mopR) of A. calcoaceticus NCIB8250 was constructed to detect phenol. P(mphK) containing three inverted repeats (IR1, IR2 and IR3) upstream of mphK was activated by the regulator MopR in the presence of phenol. Deletion analysis of P(mphK) revealed that IR2 and IR3 were essential for promoter activity, while IR1 was involved in transcriptional repression. The sensitivity of the bioreporter for the detection of phenol (0.1-5 microM) was improved by about 100% through deletion of IR1 in P(mphK).

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.

Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme. Purification and characterization of the reductase moiety.

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.

Polypeptide Requirement of Multicomponent Monooxygenase DsoABCDEF for Dimethyl Sulfide Oxidizing Activity.

In our previous study, the multicomponent monooxygenase DsoABCDEF in Acinetobacter sp. strain 20B was cloned based on its ability to oxidize dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO) in E. coli cells, which had high sequence similarity with phenol hydroxylase MopKLMNOP in Acinetobacter calcoaceticus NCIB8250, DmpKLMNOP in Pseudomonas sp. CF600 and some other multicomponent monooxygenases. In this study, DsoB, C, D, E, and F were found to be needed for DMS-oxidizing activity in polypeptide requirement experiments, while DsoA was not necessary for it. It was also found that complementation of the deletion mutants lacking DsoC or F with the corresponding Dmp polypeptides supported the DMS-oxidizing activity, while complementation of the deletion mutants lacking any of the oxygenase subunits (DsoB, D, or E) with the corresponding Dmp polypeptides reduced or nullified the activity.

The Acinetobacter calcoaceticus NCIB8250 mop operon mRNA is differentially degraded, resulting in a higher level of the 3' CatA-encoding segment than of the 5' phenolhydroxylase-encoding portion.

The 7.5-kb polycistronic mop mRNA is differentially degraded in Acinetobacter calcoaceticus. The 4.9-kb 5' portion of the transcript contains the genes mopKLMNOP, encoding the multi-component phenol hydroxylase, and its 5' end decays three times faster than the 2.3-kb 3' portion encoding catechol 1,2-dioxygenase (catA). Larger amounts of the catA mRNA than the mopKLMNOP mRNA are present in the cells as a result of this processing. The site for endonucleolytic cleavage is located in the intercistronic region between mopP and catA, and contains a potential stem-loop structure and a putative RNase E cleavage site. Decay of the mop mRNA in Escherichia coli depends on RNase E. Thus, we propose that an RNase E-like activity is also present in A. calcoaceticus. Expression of MopN, one polypeptide of the multi-component phenol hydroxylase, interferes with growth of A. calcoaceticus. Thus, harmful expression of MopN may be reduced by rapid decay of its mRNA, indicating that mRNA processing contributes to differential gene expression in the large mop operon of A. calcoaceticus NCIB8250.

Purification, biochemical properties and substrate specificity of a catechol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresistens

A catechol 1,2-dioxygenase (C1,2O) has been purified to homogeneity from Acinetobacter radioresistens grown on phenol as the sole carbon and energy source. The C1,2O appears to be a homodimer, with a molecular mass of 78 000 Da. At relatively high ionic strengths (0.5 M Na 2SO 4) subunit dissociation occurs and the monomeric unit (38 700 Da) is shown to be active. This phenomenon has never been observed before in dioxygenases. The purified C1,2O contains 0.96 iron(III) ions per unit and spectroscopic measurements suggest the presence of one high-spin iron(III) ion in an environment characteristic of intradiol cleaving enzymes. The NH 2-terminal amino acid sequence has been determined and compared to the primary structures of intradiol rings cleaving dioxygenases from other Acinetobacter strains revealing 45% homology with the benzoate-grown A. calcoaceticus ADP-1 and an identity of only one of the 20 amino acids sequenced for the phenol-grown A. calcoaceticus NCIB 8250.

Expression, inducer spectrum, domain structure, and function of MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250.

Degradation of phenol by Acinetobacter calcoaceticus NCIB8250 involves (sigma54-dependent expression of a multicomponent phenol hydroxylase and catechol 1,2-dioxygenase encoded by the mop operon. Complementation of a new mutant deficient in phenol utilization yielded the regulatory locus mopR. It is located in divergent orientation next to the mop operon. MopR is constitutively expressed at a low level from a sigma70-type promoter and belongs to the NtrC family of regulators. The amino acid sequence is similar to that of XylR regulating xylene degradation and to that of DmpR regulating dimethylphenol degradation in Pseudomonas spp. However, it shows a different effector profile for substituted phenols than DmpR. MopR activates phenol hydroxylase expression in the presence of phenol in Escherichia coli, indicating that it binds the effector. The phenol binding A domains of MopR and DmpR have fewer identical residues than the A domains of DmpR and XylR, despite the fact that XylR recognizes different effectors. This suggests that sequence conservation in the A domain does not reflect the potential to bind the respective effectors. Overexpression of the MopR A domain in the presence of wild-type MopR causes loss of mop inducibility by phenol, establishing its negative transdominance over MopR. Deletion of 110 residues from the N terminus did not affect transdominance of the truncated domain, whereas deletion of 150 residues abolished it completely. This result establishes the distinction of two subdomains, A(N) and A(C), which together constitute the A domain. The C-terminal portion of the A domain, A(C), shows considerable affinity for the C domain, even in the presence of the trigger phenol.

Genetic organization, nucleotide sequence and regulation of expression of genes encoding phenol hydroxylase and catechol 1,2‐dioxygenase in Acinetobacter calcoaceticus NCIB8250

We have mutated Acinetobacter calcoaceticus NCIB-8250 to growth deficiency on phenol as sole carbon source and isolated genes with similarity to phenol hydroxylase and catechol 1,2-dioxygenase by complementation. Sequence analysis reveals the presence of six open reading frames (ORFs) with similarities to a Pseudomonas multicomponent phenol hydroxylase which are followed by an ORF with similarity to catA from A. calcoaceticus ADP1. Transformation of these genes to ADP1 confers the ability to grow at the expense of phenol as sole carbon source. Primer extension analysis indicates phenol-inducible transcription from an RpoN-dependent promoter sharing sequence similarity with the sigma 54 consensus promoter sequence, except that the -12 box is GG instead of GC. A catA::lacZ transcriptional fusion shows the same induction profile for beta-galactosidase expression as transcription from the sigma 54-dependent promoter. This result suggests that catA is cotranscribed in the same operon with the phenol hydroxylase-encoding genes and is consistent with the fact that no apparent additional promoter is found for catA by sequence analysis or primer extension. Catechol 1,2-dioxygenase activity is induced in NCIB8250 by benzoate, whereas beta-galactosidase expression from the catA::lacZ fusion is not. This observation leads to the hypothesis that two differentially regulated catA genes should be present in that strain.

Phenol degradation by Acinetobacter calcoaceticus NCIB 8250

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.

Microbial Baeyer-Villiger reaction of bicyclo[3.2.0]heptan-6-ones — a novel approach to sarkomycin A

Racemic (1α,2α,5α)- and (1β,2α,5β)-2-bromobicyclo[3.2.0]heptan-6-one ( rac- 7, rac- 10, respectively), (1α,2α,5β)- and (1β,2α,5β)-2-benzyloxybicyclo[3.2.0]heptan-6-one ( rac- 15, rac- 13, respectively), (1β,2α,5β)-2-hydroxy ( rac- 17) and cis-bicyclo[3.2.0]hept-2-en-7-one ( rac- 18) were subjected to a microbial Baeyer-Villiger reaction by Acinetobacter calcoaceticus NCIB 9871. In each case both regioisomeric lactones were formed (67–93 % yield) having always the opposite configuration (20 to > 99 % e.e.). Both the ratio of the regioisomers and the enantiomeric excess proved to be dependent on the type of substitution. Analogously cis-bicyclo[3.2.0.]heptan-2,6-dione ( rac- 1) gave besides other products cyclosarkomycin ( 1b) (7 % yield, 97 % e.e.). Compound 1b was also obtained from the Baeyer-Villiger product of rac- 17 by Swern oxidation (total yield starting from rac- 17 9 %, > 98 % e.e.).

Asymmetric microbiological baeyer-villiger reaction of a bridged bicyclic ketone: divergent behaviour of its enantiomers

The biconversion of 7-anti-benzyloxymethylbicyclo[2.2.1]hept-5-en-2-one 1 with cells of Acinetobacter calcoaceticus NCIB 9871 has been studied. Its (1R,4S,7R) enantiomer underwent a Baeyer-Villiger oxidation yielding rearranged lactone 2 (ee 85%), whereas (1S,4R,7S)- 1 was reduced to alcohols 3 -endo (ee 95%) and 3 -exo (o.p. 89%). Biotransformation of rac- 1 by Acinetobacter calcoaceticus yielded the rearranged lactone (1 S)- 2 from (1 R)- 1 and the alcohols 3 from (1 S)- 1 .

Multiple forms of carboxylesterase activity inAcinetobacter calcoaceticus

A carboxylesterase activity (E.C.3.1.1) was found in all four strains of Acinetobacter calcoaceticus tested. The activity was present in both 2 X 10(4) gav h-1 supernatant and bacterial wall-membrane fractions. The activity in the supernatant was in two molecular weight forms, the predominant form with a Mr of about 10(3) K and a minor form Mr approximately 600 K. The activity was inhibited by phenylmethylsulfonyl fluoride. SDS-PAGE showed that in A. calcoaceticus NCIB 8250 the activity was composed of three components of Mr 43, 40 and 38 K. The individual components showed different activities with 1- or 2-naphthyl esters. Of the other strains used, one showed a nearly identical pattern of component activities, while the other two showed only two component activities.

Multiple forms of carboxylesterase activity in Acinetobacter calcoaceticus.

A carboxylesterase activity (E.C.3.1.1) was found in all four strains of Acinetobacter calcoaceticus tested. The activity was present in both 2 X 10(4) gav h-1 supernatant and bacterial wall-membrane fractions. The activity in the supernatant was in two molecular weight forms, the predominant form with a Mr of about 10(3) K and a minor form Mr approximately 600 K. The activity was inhibited by phenylmethylsulfonyl fluoride. SDS-PAGE showed that in A. calcoaceticus NCIB 8250 the activity was composed of three components of Mr 43, 40 and 38 K. The individual components showed different activities with 1- or 2-naphthyl esters. Of the other strains used, one showed a nearly identical pattern of component activities, while the other two showed only two component activities.

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus

1. Acinetobacter calcoaceticus NCIB 8250 can grow on benzyl alcohol as sole source of carbon and energy. Previous work had shown that benzyl alcohol is oxidised to benzoate by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II. This thesis is concerned with the purification and characterisation of these two NAD+-dependent, soluble enzymes.

Structure, Distribution and Function of Wax Esters in Acinetobacter calcoaceticus

SUMMARY: The wax esters of Acinetobacter calcoaceticus strains NCIB 8250 and NCIB 10487 harvested at stationary phase from N-limited batch cultures were extracted and shown to consist of C14 to C18 saturated and mono-unsaturated alkan-l-ols randomly esterified with C14 to C18 saturated and mono-unsaturated fatty acids. The mono-unsaturated components contained a cis Δ9 double bond. Wax ester content of strain NCIB 8250 increased under conditions of low growth rate in N-limited continuous culture with carbon and energy source in excess. The high content of wax ester in N-limited cultures of strain NCIB 8250 was lowered by incubation in the absence of a carbon and energy source and the wax ester was converted to water-soluble materials and CO2. It is proposed that in A. calcoaceticus NCIB 8250 the endogenous wax ester present in N-limited cells can serve as an energy reserve. All 19 strains of A. calcoaceticus tested contained some wax ester and as 16 of these strains had increased wax ester contents when harvested from stationary phase N-limited batch cultures, it appears that wax esters are widespread, but not universal, energy storage components in the genus Acinetobacter.

The interaction of phosphonium ions with Acinetobacter calcoaceticus: evidence for the operation of an efflux system

Experiments with washed cell suspensions of Acinetobacter calcoaceticus NCIB 8250 indicated that this organism possesses an energy-linked efflux system for phosphonium ions and related compounds. Evidence is presented to show that the apparent impermeability to low concentrations of methyltriphenylphosphonium ion is a result of the combination of the outer membrane acting as a partial permeability barrier and the activity of the efflux system.

Growth Yields and Respiratory Efficiency of Acinetobacter calcoaceticus

SUMMARY: Acinetobacter calcoaceticus NCIB 8250 was grown in batch culture on 46 separate sources of carbon + energy and the molar growth yields were measured. The organism was also grown in continuous culture on L-mandelate, phenylglyoxylate and succinate and the results used to calculate maximum molar growth yields. The elemental composition of the bacteria was determined. Growth equations for each substrate were constructed so that the amount of oxygen consumed per mol of substrate could be calculated. Growth yields on oxygen (Yo2) were then estimated. The Yo2 values were low [averaging about 19 g dry wt (mol O2 -1]. The most likely explanation for the low yields is that the effective P/O ratio is only about one, although there are probably at least two sites of oxidative phosphorylation.

Effect of Oxygen Concentration on the Growth and Respiratory Efficiency of Acinetobacter calcoaceticus

Maximum molar growth yields on oxygen ( Y O 2 max ) and succinate ( Y succinate max ) were not affected when carbon-limited Acinetobacter calcoaceticus NCIB 8250 was changed to oxygen limitation.→H+/O quotients for cells grown with succinate- or ammonium-limitation did not differ significantly from those obtained with oxygen-limited cells. The levels of cytochromes b and o remained constant at 0‧13 and 0‧05 nmol (mg protein)–1 respectively in crude cell-free extracts of succinate-limited organisms grown at high (80 mmHg) or low (5–10 mmHg) dissolved oxygen tension respectively. Their levels increased in oxygen-limited conditions, when cytochrome a 2(d) was additionally synthesized and organisms exhibited increased resistance to cyanide inhibition of respiration. It is concluded that the respiratory efficiency of A. calcoaceticus NCIB 8250 is unaffected by oxygen concentration under the conditions studied. Theoretical calculations are presented for maximum molar growth yields on succinate and oxygen and these are compared with experimental values. Although the effective P/O ratio is only about unity, energy is probably conserved at two sites of oxidative phosphorylation and this is independent of the de novo synthesis of cytochrome a 2 (d) under conditions of oxygen limitation. Possible reasons for the observed low molar growth yields are discussed.

Gene transfer in Acinetobacter calcoaceticus NCIB8250

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.