Discovery of a novel periplasmic protein that forms a complex with a trimeric autotransporter adhesin and peptidoglycan

Trimeric autotransporter adhesins (TAAs), fibrous proteins on the cell surface of Gram‐negative bacteria, have attracted attention as virulence factors. However, little is known about the mechanism of their biogenesis. AtaA, a TAA of Acinetobacter sp. Tol 5, confers nonspecific, high adhesiveness to bacterial cells. We identified a new gene, tpgA, which forms a single operon with ataA and encodes a protein comprising two conserved protein domains identified by Pfam: an N‐terminal SmpA/OmlA domain and a C‐terminal OmpA_C‐like domain with a peptidoglycan (PGN)‐binding motif. Cell fractionation and a pull‐down assay showed that TpgA forms a complex with AtaA, anchoring it to the outer membrane (OM). Isolation of total PGN‐associated proteins showed TpgA binding to PGN. Disruption of tpgA significantly decreased the adhesiveness of Tol 5 because of a decrease in surface‐displayed AtaA, suggesting TpgA involvement in AtaA secretion. This is reminiscent of SadB, which functions as a specific chaperone for SadA, a TAA in Salmonella species; however, SadB anchors to the inner membrane, whereas TpgA anchors to the OM through AtaA. The genetic organization encoding the TAA–TpgA‐like protein cassette can be found in diverse Gram‐negative bacteria, suggesting a common contribution of TpgA homologues to TAA biogenesis.     

Effect of Cell Appendages on the Adhesion Properties of a Highly Adhesive Bacterium,Acinetobacter sp. Tol 5

A toluene-degrading bacterium, Acinetobacter sp. Tol 5, shows noteworthy adhesiveness mediated by two types of cell appendages. In this study, we obtained a less-adhesive mutant, T1, which lost both types of appendages, and investigated how the cell appendages affect the adhesion properties of this useful bacterium for environmental technology. Wild-type cells attained irreversible adhesion to polyurethane carriers within 30 s, while adhesion of T1 cells was still reversible at that time. While T1 showed decreased adhesion with decreasing ionic strength and did not adhere at all at 0.015 mM, adhesion of the wild type was fully independent of ionic strength. Acinetobacter sp. Tol 5 was also found to be not motile. Our results suggest that through the long distant interaction mediated by the appendages between the cells and surfaces, Tol 5 cells can attain irreversible adhesion very quickly without approaching the vicinity of the substratum.     

On-fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin

In the cell surface display system, the distance of a surface-displayed molecule from the cell surface should influence its functionality due to the interference by other surface structures. For the purpose of developing this distance-variable surface display system, we utilized a long fibrous adhesin, Acinetobacter trimeric autotransporter adhesin (AtaA) of the strain Tol 5. We constructed His-tagged full-length and shorter AtaA fibers designed by N-terminal deletion and expressed them in the ΔataA mutant. Immunoelectron microscopy clearly showed that they formed fibers on the cell surface and the His-tag was displayed on the fiber tip located at fixed distances from the cell surface. N-terminal deletion of AtaA shortened the distance between the His-tag and the cell surface, as designed. Time-course analyses of the cell-to-Ni-Sepharose beads binding revealed that cells producing the longer fibers bound more rapidly to the beads. The His-tagged AtaA derivatives were also displayed on Escherichia coli cells, and a similar tendency was shown; the His-tag on the longer fiber was more functional than that on the shorter one. Thus, we developed an on-fiber display system of a functional peptide using a long trimeric autotransporter adhesin (TAA) fiber, which can vary the distance between the displayed molecule and the cell surface.     

Two Morphological Types of Cell Appendages on a Strongly Adhesive Bacterium, Acinetobacter sp. Strain Tol 5

Two morphological types of appendages, an anchor-like appendage and a peritrichate fibril-type appendage, have been observed on cells of an adhesive bacterium, Acinetobacter sp. strain Tol 5, by use of recently developed electron microscopic techniques. The anchor extends straight to the substratum without branching and tethers the cell body at its end at distances of several hundred nanometers, whereas the peritrichate fibril attaches to the substratum in multiple places, fixing the cell at much shorter distances.     

Effect of cell appendages on the adhesion properties of a highly adhesive bacterium, Acinetobacter sp. Tol 5

A toluene-degrading bacterium, Acinetobacter sp. Tol 5, shows noteworthy adhesiveness mediated by two types of cell appendages. In this study, we obtained a less-adhesive mutant, T1, which lost both types of appendages, and investigated how the cell appendages affect the adhesion properties of this useful bacterium for environmental technology. Wild-type cells attained irreversible adhesion to polyurethane carriers within 30 s, while adhesion of T1 cells was still reversible at that time. While T1 showed decreased adhesion with decreasing ionic strength and did not adhere at all at 0.015 mM, adhesion of the wild type was fully independent of ionic strength. Acinetobacter sp. Tol 5 was also found to be not motile. Our results suggest that through the long distant interaction mediated by the appendages between the cells and surfaces, Tol 5 cells can attain irreversible adhesion very quickly without approaching the vicinity of the substratum.     

Development of Spatial Distribution Patterns by Biofilm Cells

Confined spatial patterns of microbial distribution are prevalent in nature, such as in microbial mats, soil communities, and water stream biofilms. The symbiotic two-species consortium of Pseudomonas putida and Acinetobacter sp. strain C6, originally isolated from a creosote-polluted aquifer, has evolved a distinct spatial organization in the laboratory that is characterized by an increased fitness and productivity. In this consortium, P. putida is reliant on microcolonies formed by Acinetobacter sp. C6, to which it attaches. Here we describe the processes that lead to the microcolony pattern by Acinetobacter sp. C6. Ecological spatial pattern analyses revealed that the microcolonies were not entirely randomly distributed and instead were arranged in a uniform pattern. Detailed time-lapse confocal microscopy at the single-cell level demonstrated that the spatial pattern was the result of an intriguing self-organization: small multicellular clusters moved along the surface to fuse with one another to form microcolonies. This active distribution capability was dependent on environmental factors (carbon source and oxygen) and historical contingency (formation of phenotypic variants). The findings of this study are discussed in the context of species distribution patterns observed in macroecology, and we summarize observations about the processes involved in coadaptation between P. putida and Acinetobacter sp. C6. Our results contribute to an understanding of spatial species distribution patterns as they are observed in nature, as well as the ecology of engineered communities that have the potential for enhanced and sustainable bioprocessing capacity.     

Heterologous expression of geraniol dehydrogenase for identifying the metabolic pathways involved in the biotransformation of citral by Acinetobacter sp. Tol 5

ABSTRACT

The biotransformation of citral, an industrially important monoterpenoid, has been extensively studied using many microbial biocatalysts. However, the metabolic pathways involved in its biotransformation are still unclear, because citral is a mixture of the trans-isomer geranial and the cis-isomer neral. Here, we applied the heterologous expression of geoA, a gene encoding geraniol dehydrogenase that specifically converts geraniol to geranial and nerol to neral, to identify the metabolic pathways involved in the biotransformation of citral. Acinetobacter sp. Tol 5 was employed in order to demonstrate the utility of this methodology. Tol 5 transformed citral to (1R,3R,4R)-1-methyl-4-(1-methylethenyl)-1,3-cyclohexanediol and geranic acid. Biotransformation of citral precursors (geraniol and nerol) by Tol 5 transformant cells expressing geoA revealed that these compounds were transformed specifically from geranial. Our methodology is expected to facilitate a better understanding of the metabolic pathways involved in the biotransformation of substrates that are unstable and include geometric isomers.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

Engineering the Genotype of Acinetobacter sp. Strain ADP1 To Enhance Biosynthesis of Cyanophycin

To study the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophycin (CGP) in Acinetobacter sp. strain ADP1, genes encoding the putative arginine regulatory protein (argR) and the arginine succinyltransferase (astA) were inactivated, and the effects of these mutations on CGP synthesis were analyzed. The inactivation of these genes resulted in a 3.5- or 7-fold increase in CGP content, respectively, when the cells were grown on glutamate. Knockout mutations in both genes led to a better understanding of the effect of the addition of other substrates to arginine on CGP synthesis during growth of the cells of Acinetobacter sp. strain ADP1. Overexpression of ArgF (ornithine carbamoyltransferase), CarA-CarB (small and large subunits of carbamoylphosphate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohydrate metabolism, showed a significant increase in CGP contents when grown on mineral medium supplemented with glutamate, aspartate, or arginine. The Acinetobacter sp. ΔastA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon. Phosphate starvation is the only known trigger for CGP synthesis in this bacterium, which possesses the PhoB/PhoR phosphate regulon system. Overexpression of phoB caused an 8.6-fold increase in CGP content in comparison to the parent strain at a nonlimiting phosphate concentration.     

Enrichment for Hydrogen-Oxidizing Acinetobacter spp. in the Rhizosphere of Hydrogen-Evolving Soybean Root Nodules

Field soybean plants were inoculated with Hup⁺ wild-type or H₂ uptake-negative (Hup⁻) mutants of Bradyrhizobium japonicum. For two consecutive summers we found an enrichment for acinetobacters associated with the surfaces of the H₂-evolving nodules. Soybean root nodules that evolved H₂ had up to 12 times more Acinetobacter spp. bacteria associated with their surfaces than did nodules incapable of evolving H₂. All of the newly isolated strains identified as Acinetobacter obtained from the surfaces of root nodules, as well as known established Acinetobacter strains, were capable of oxidizing H₂, a property not previously described for this alkane-degrading soil bacterium.     

Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of Acinetobacter sp. Strain ADP1

Hydroxycinnamates, aromatic compounds that play diverse roles in plants, are dissimilated by enzymes encoded by the hca genes in the nutritionally versatile, naturally transformable bacterium Acinetobacter sp. strain ADP1. A key step in the hca-encoded pathway is activation of the natural substrates caffeate, p-coumarate, and ferulate by an acyl:coenzyme A (acyl:CoA) ligase encoded by hcaC. As described in this paper, Acinetobacter cells with a knockout of the next enzyme in the pathway, hydroxycinnamoyl-CoA hydratase/lyase (HcaA), are extremely sensitive to the presence of the three natural hydroxycinnamate substrates; Escherichia coli cells carrying a subclone with the hcaC gene are hydroxycinnamate sensitive as well. When the hcaA mutation was combined with a mutation in the repressor HcaR, exposure of the doubly mutated Acinetobacter cells to caffeate, p-coumarate, or ferulate at 10⁻⁶ M totally inhibited the growth of cells. The toxicity of p-coumarate and ferulate to a ΔhcaA strain was found to be a bacteriostatic effect. Although not toxic to wild-type cells initially, the diphenolic caffeate was itself converted to a toxin over time in the absence of cells; the converted toxin was bactericidal. In an Acinetobacter strain blocked in hcaA, a secondary mutation in the ligase (HcaC) suppresses the toxic effect. Analysis of suppression due to the mutation of hcaC led to the development of a positive-selection strategy that targets mutations blocking HcaC. An hcaC mutation from one isolate was characterized and was found to result in the substitution of an amino acid that is conserved in a functionally characterized homolog of HcaC.     

Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of Acinetobacter sp. Strain ADP1

The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5′His₆WS/DGAT comprising an N-terminal His₆ tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5′His₆atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein⁻¹ min⁻¹ was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5′His₆WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1ΩKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1ΩKm in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.     

Complete Genome Sequence of the Diesel-Degrading Acinetobacter sp. Strain DR1

The genus Acinetobacter is ubiquitous in soil, aquatic, and sediment environments and includes pathogenic strains, such as A. baumannii. Many Acinetobacter species isolated from various environments have biotechnological potential since they are capable of degrading a variety of pollutants. Acinetobacter sp. strain DR1 has been identified as a diesel degrader. Here we report the complete genome sequence of Acinetobacter sp. DR1 isolated from the soil of a rice paddy.The genus Acinetobacter appears to be metabolically versatile and has the ability to degrade aliphatic hydrocarbon, thus making it an organism of interest for its possible bioremediational potential (9). Despite its biotechnological potential, the majority of genome projects conducted with Acinetobacter species have focused on pathogenic strains of A. baumannii. Currently, the only available whole-genome sequence of environmental isolates is that of A. baylyi ADP1 (2). Acinetobacter sp. strain DR1 was isolated from the soil of rice paddies, located in Deok-So (Korea University Agricultural Station), in the Kyonggi province of South Korea. Strain DR1 is capable of utilizing aliphatic hydrocarbons and diesel oil (5). Similarly to A. baylyi ADP1, this strain is also competent for natural transformation. We demonstrated previously that sodium chloride added to the medium induces the overproduction of exopolysaccharide (EPS), which evidences protective activity against diesel toxicity (4). Interestingly, DR1 possesses a quorum sensing (QS) system, which has been shown to play a significant role in biofilm formation and hexadecane biodegradation. The results of proteomic studies have demonstrated that the QS system regulates a broad variety of proteins (6). Collectively, our findings demonstrate that DR1 has profound potential for environmental applications and is an environmental isolate distinct from pathogenic strains, thus indicating that the whole-genome sequencing of DR1 is a worthwhile pursuit.Initial pyrosequencing using a GS-FLX system (454 Life Science Corporation) generated 652,162 reads (264,482,836 nucleotides; 64.3-fold coverage), which were assembled into 56 contigs. To determine the order of the contigs, 1,248 fosmid clones were constructed with an average insert size of 35 kb (10.5-fold coverage). The fosmid-end sequencing of 936 clones generated 1,372,452 bp. These high-quality Sanger reads allowed the assembly of 41 large contigs into 2 scaffolds containing 38 gaps. The gaps were filled via primer walking. All procedures for genome sequencing and gap filling were conducted by Macrogen (Seoul, South Korea). Protein coding regions were predicted with the GLIMMER3 software program (3), and automatic genome annotation was conducted on a RAST server (1) and the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). The tRNA and rRNA genes were annotated using the tRNAScan-SE (8) and RNAmmer software programs (7), respectively. The genome of Acinetobacter sp. DR1 consists of a circular 4,152,543-bp chromosome with a G+C content of 38%, 3,874 predicted coding sequences, and 71 tRNAs. There are 6 rRNA operons with a 16S, tRNA-Ile, tRNA-Ala, 23S, 5S organization. The genes studied previously were clearly identified via genome sequencing (4, 5, 6). The availability of the complete genome sequence of Acinetobacter sp. strain DR1 will contribute to an in-depth understanding of the genetic potentials of Acinetobacter species.     

Characterization of Two Novel Propachlor Degradation Pathways in Two Species of Soil Bacteria

Propachlor (2-chloro-N-isopropylacetanilide) is an acetamide herbicide used in preemergence. In this study, we isolated and characterized a soil bacterium, Acinetobacter strain BEM2, that was able to utilize this herbicide as the sole and limiting carbon source. Identification of the intermediates of propachlor degradation by this strain and characterization of new metabolites in the degradation of propachlor by a previously reported strain of Pseudomonas (PEM1) support two different propachlor degradation pathways. Washed-cell suspensions of strain PEM1 with propachlor accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain PEM1 grew on propachlor with a generation time of 3.4 h and a K_s of 0.17 ± 0.04 mM. Acinetobacter strain BEM2 grew on propachlor with a generation time of 3.1 h and a K_s of 0.3 ± 0.07 mM. Incubations with strain BEM2 resulted in accumulation of N-isopropylacetanilide, N-isopropylaniline, isopropylamine, and catechol. Both degradative pathways were inducible, and the principal product of the carbon atoms in the propachlor ring was carbon dioxide. These results and biodegradation experiments with the identified metabolites indicate that metabolism of propachlor by Pseudomonas sp. strain PEM1 proceeds through a different pathway from metabolism by Acinetobacter sp. strain BEM2.     

Cloning and Genetic Characterization of dca Genes Required for β-Oxidation of Straight-Chain Dicarboxylic Acids in Acinetobacter sp. Strain ADP1

A previous study of deletions in the protocatechuate (pca) region of the Acinetobacter sp. strain ADP1 chromosome revealed that genes required for utilization of the six-carbon dicarboxylic acid, adipic acid, are linked to the pca structural genes. To investigate the genes involved in adipate catabolism, a 33.8-kb SacI fragment, which corrects a deletion spanning this region, was cloned. In addition to containing known pca, qui, and pob genes (for protocatechuate, quinate, and 4-hydroxybenzoate dissimilation), clone pZR8000 contained 10 kb of DNA which was the subject of this investigation. A mutant strain of Escherichia coli DH5α, strain EDP1, was isolated that was able to utilize protocatechuate and 4-hydroxybenzoate as growth substrates when EDP1 cells contained pZR8000. Sequence analysis of the new region of DNA on pZR8000 revealed open reading frames predicted to be involved in β-oxidation. Knockouts of three genes implicated in β-oxidation steps were introduced into the chromosome of Acinetobacter sp. strain ADP1. Each of the mutants was unable to grow with adipate. Because the mutants were affected in their ability to utilize additional saturated, straight-chain dicarboxylic acids, the newly discovered 10 kb of DNA was termed the dca (dicarboxylic acid) region. Mutant strains included one with a deletion in dcaA (encoding an acyl coenzyme A [acyl-CoA] dehydrogenase homolog), one with a deletion in dcaE (encoding an enoyl-CoA hydratase homolog), and one with a deletion in dcaH (encoding a hydroxyacyl-CoA dehydrogenase homolog). Data on the dca region should help us probe the functional significance and interrelationships of clustered genetic elements in this section of the Acinetobacter chromosome.     

Silica-immobilized Methylobacterium sp. NP3 and Acinetobacter sp. PK1 degrade high concentrations of phenol

Aims: To immobilize Methylobacterium sp. NP3 and Acinetobacter sp. PK1 to silica and determine the ability of the immobilized bacteria to degrade high concentrations of phenol. Methods and Results: The phenol degradation activity of suspended and immobilized Methylobacterium sp. NP3 and Acinetobacter sp. PK1 bacteria was investigated in batch experiments with various concentrations of phenol. The bacterial cells were immobilized by attachment to or encapsulation in silica. The encapsulated bacteria had the highest phenol degradation rate, especially at initial phenol concentrations between 7500 and 10 000 mg l^?1. Additionally, the immobilized cells could continuously degrade phenol for up to 55 days. Conclusions: The encapsulation of a mixed culture of Methylobacterium sp. NP3 and Acinetobacter sp. PK1 is an effective and easy technique that can be used to improve bacterial stability and phenol degradation. Significance and Impact of the Study: Wastewater from various industries contains high concentrations of phenol, which can cause wastewater treatment failure. Silica‐immobilized bacteria could be applied in bioreactors to initially remove the phenol, thereby preventing phenol shock loads to the wastewater treatment system.     

Transformation by Polyoma Virus affects Adhesion of Fibroblasts

WHEN freshly trypsinized hamster fibroblasts (BHK 21, Clone 13¹) are shaken in suspension, intercellular adhesions are rapidly formed and there is some support for the view that this process is the expression of an adhesive property of the cells in culture which has survived the procedures used to separate them and is not simply an artefact of the use of trypsin². Transformation of animal cells in culture by tumour viruses alters the cell surfaces, as various techniques have shown^3–9 and we have investigated whether intercellular adhesiveness, as defined by the particular assay we have been using, is also affected.     

Cell surface of the fish pathogenic bacterium Aeromonas salmonicida: II. Purification and characterization of a major cell envelope protein related to autoagglutination,adhesion and virulence

The purification of the major protein of the membrane fraction of an autoagglutinating strain of Aeromonas salmonicida is described. This protein, designated as additional cell envelope protein, is water-insoluble, has a molecular weight of about 54 000 and its amino terminal sequence is H₂N-Asp-Val-Leu-Leu. Neither sulphur-containing amino acids nor sugar residues were detected. Its amino acid composition, which shows that the additional cell envelope protein is hydrophobic in nature, is remarkably similar to those of various proteins known to be present in additional surface layers of other bacteria, to the adhesive K88 fimbriae of enteropathogenic Escherichia coli and to a pore protein of the outer membrane of E. coli K12.     

Transformation of Acinetobacter sp. Strain BD413(pFG4ΔnptII) with Transgenic Plant DNA in Soil Microcosms and Effects of Kanamycin on Selection of Transformants

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 10⁹ CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.     

Long-Chain Aldehyde Dehydrogenase That Participates in n-Alkane Utilization and Wax Ester Synthesis in Acinetobacter sp. Strain M-1

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C₄ to C₁₄), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Δ) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.