Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a Proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5 kDa) and 347 aa (38.6 kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

Alternate hydrophobic sites on the cell surface of Acinetobacter calcoaceticus RAG-1

Abstract Mutants of A. calcoaceticus RAG-1 lacking thin fimbriae (35 Å) do not adhere to hydrophobic surfaces [5], or grow on hydrocarbons under conditions of weak agitation and small inocula. Emulsan-deficient derivatives of such mutants, isolated in the present study, (i) lacked cell-surface emulsan, (ii) adhered avidly to hydrocarbons, (iii) lacked thin fimbriae, and (iv) regained the capacity to grow on hydrocarbons. The results show that emulsan masks an alternate hydrophobic site(s) on the cell surface of RAG-1.     

Relationship between emulsifying activity and carbohydrate backbone structure of emulsan from Acinetobacter calcoaceticus RAG-1

Various emulsan samples with the different degrees of branching of the carbohydrate backbone were obtained from Acinetobacter calcoaceticus under different culture conditions. The emulsifying activity of emulsan had a linear correlation to the branching degrees of the carbohydrate backbone (r²= 0.930) suggesting that the structure of carbohydrate backbone was an important factor influencing emulsifying activity. This revised version was published online in November 2006 with corrections to the Cover Date.     

Cloning of the genes involved in synthesis of coenzyme pyrrolo-quinoline-quinone from Acinetobacter calcoaceticus.

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.     

Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene

The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic-interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long acyl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg-yolk emulsions. The N-terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signai sequence at the N-terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two-step translocation mechanism. However, no chaperone-encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disutphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary-structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site-directed mutagenesis, resulting in production of an inactive extracellular lipase.     

Cloning and sequencing of genes encoding malonate decarboxylase in Acinetobacter calcoaceticus

Malonate decarboxylase from Acinetobacter calcoaceticus was isolated and characterized (Kim, Y.S., Byun, H.S., J. Biol. Chem. 269 (1994) 29636–29641), and its subunits were reanalyzed recently to be α, β, γ, and δ. The genes for the subunits, MdcA (548 a.a.), B (295 a.a.), C (238 a.a.), and D (102 a.a.), of the enzyme have been cloned by using oligonucleotide primers deduced from amino acid sequences of peptides isolated from the purified enzyme, and sequenced to be clustered in an operon in the order of A-D-B-C. The operon was found to encode more genes than mdcABCD. The Escherichia coli, transformed with the vector containing the insert mdcADBC and about 1.7 kb of an upstream region, expressed the four subunits of the enzyme but the proteins did not show enzyme activity. It indicates that, like the enzymes from Malonomonas rubra and Klebsiella pneumoniae, more genes are needed for the formation of the functional malonate decarboxylase.     

Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane

Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen.     

Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1.

An extracellular lipase, LipA, extracted from Acinetobacter sp. RAG-1 grown on hexadecane was purified and properties of the enzyme investigated. The enzyme is released into the growth medium during the transition to stationary phase. The lipase was harvested from cells grown to stationary phase, and purified with 22% yield and > 10-fold purification. The protein demonstrates little affinity for anion exchange resins, with contaminating proteins removed by passing crude supernatants over a Mono Q column. The lipase was bound to a butyl Sepharose column and eluted in a Triton X-100 gradient. The molecular mass (33 kDa) was determined employing SDS/PAGE. LipA was found to be stable at pH 5.8-9.0, with optimal activity at 9.0. The lipase remained active at temperatures up to 70 degrees C, with maximal activity observed at 55 degrees C. LipA is active against a wide range of fatty acid esters of p-nitrophenyl, but preferentially attacks medium length acyl chains (C6, C8). The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides, as demonstrated by zymogram analysis. RAG-1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+. The lipase is strongly inhibited by EDTA, Hg2+, and Cu2+, but shows no loss in activity after incubation with other metals or inhibitors examined in this study. The protein retains more than 75% of its initial activity after exposure to organic solvents, but is rapidly deactivated by pyridine. RAG-1 lipase offers potential for use as a biocatalyst.     

Purification and characterization of lipase from psychrophilic Acinetobacter calcoaceticus LP009

A lipase-producing bacterium, Acinetobacter calcoacetius LP009, was isolated from raw milk. The optimum conditions for growth and lipase production by A. calcoaceticus LP009 were 15 degrees C with shaking at 200 rpm in LB supplemented with 1.0% (v/v) Tween 80. The crude lipase was purified to homogeneous state by ultrafiltration and gel filtration chromatography on Sephadex G-100. Its molecular weight determined by SDS-PAGE was 23 kDa and it exhibited maximum activity at pH 7.0 and 50 degrees C. It was stable over the pH range of 4.0 to 8.0 and at temperatures lower than 45 degrees C. It was a metalloenzyme that is positionally non-specific and had the ability to improve fat hydrolysis in soybean meal and in premixed animals feed.     

Relationship between phage resistance and emulsan production,interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1

The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 10⁶) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain.     

Cloning a sequence, coding an SKT1 family protease inhibitor from potato

Seven structurally similar clones from potato (Solanum tuberosum L.), cv. Istrinskii genomic DNA were isolated by cloning of the PCR products. It was suggested that five of these clones were the amplified copies of the same gene. Based on comparative and structural analysis of these clones, initial nucleotide structure of the gene was reconstructed. It appeared to be highly homologous (98%) to the already published sequences encoding the proteins belonging to the soybean Kunitz trypsin inhibitor family (SKTI). Comparison of the results with the previously published data on the SKTI-type proteinase inhibitors from potato of cv. Istrinskii suggests that the gene examined encodes both chains of the earlier described PSPI-21-6.3 protein [9].     

Cloning and functional analysis of molecular chaperone genes from Bacillus stearothermophilus SIC1

Genes homologous to groES and groEL, which are recognized as molecular chaperone genes, from Bacillus stearothermophilus SIC1 were cloned and sequenced. By addition of GroES, GroEL and ATP in vitro, remaning activity of the alcohol dehydrogenase from Saccharomyces cerevisiae after heat treatment at 50°C for 6 min was improved from 55% to 90%. Furthermore, even though inclusion bodies were formed when a single chain Fv(sFv) was expressed in E. coli cells, during in vivo coexpression with molecular chaperone, a significant amount of the antibody protein could be recovered from the soluble fraction.     

Genome sequence of Acinetobacter calcoaceticus PHEA-2, isolated from industry wastewater

Genome analysis of Acinetobacter calcoaceticus PHEA-2 was undertaken because of the importance of this bacterium for bioremediation of phenol-polluted water and because of the close phylogenetic relationship of this species with the human pathogen Acinetobacter baumannii. To our knowledge, this is the first strain of A. calcoaceticus whose genome has been sequenced.     

Tolerance of Acinetobacter calcoaceticus RAG-1 to the cationic surfactant cetyltrimethylammonium bromide: role of the bioemulsifier emulsan

Emulsan, the polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1, was found to enhance the tolerance of RAG-1 cells to the toxic effects of the cationic detergent cetyltrimethylammonium bromide (CTAB). Emulsan-mediated tolerance was obtained with the purified deproteinated apoemulsan; ca. 9 micrograms of apoemulsan neutralized 1 microgram of CTAB. Deesterified apoemulsan was only half as effective in protecting the cells from CTAB toxicity. Tolerance was also mediated by the cell-associated emulsan minicapsule. Mutants lacking this capsule were more sensitive to CTAB than the corresponding parent. The growth of mutants and parent cells in mixed-culture experiments demonstrated that the cell-associated polymer mediates CTAB tolerance in the early stages of growth. Once sufficient cell-free polymer has been released into the aqueous medium (ca. 0.5 micrograms/ml), this extracellular emulsan also plays a role in CTAB tolerance.     

Cloning and sequence analysis of endoglucanase genes from an industrial fungus, Aspergillus kawachii

Three endoglucanase genes (cel5A, cel5B, and cel61A) were cloned from an industrial fungus, Aspergillus kawachii. Yeasts transformed with these cDNAs showed endoglucanase activity in medium. Cel5A and Cel61A contained a type 1 cellulose-binding domain (CBD1) at the C-terminus of the enzyme. The putative catalytic regions of Cel5A and Cel5B showed homology with various endoglucanases belonging glycosyl hydrolase family 5 (GH5). Cel5B showed high homology with Cel5A in catalytic region, but it lacked CBD1 and linker. The cel5A contained four introns, whereas cel5B contained five introns. The putative catalytic region of Cel61A showed homology with enzymes belonging to GH61. The cel61A contained no introns.