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.     

AtaA,a New Member of the Trimeric Autotransporter Adhesins from Acinetobacter sp. Tol 5 Mediating High Adhesiveness to Various Abiotic Surfaces

Acinetobacter sp. Tol 5 exhibits an autoagglutinating nature and noteworthy adhesiveness to various abiotic surfaces from hydrophobic plastics to hydrophilic glass and stainless steel. Although previous studies have suggested that bacterionanofibers on Tol 5 cells are involved in the adhesive phenotype of Tol 5, the fiber that directly mediates Tol 5 adhesion has remained unknown. Here, we present a new member of trimeric autotransporter adhesins designated AtaA, which we discovered by analyzing a less adhesive mutant of Tol 5, T1, obtained by transposon mutagenesis. AtaA forms thinner and shorter nanofibers than fimbriae on Tol 5 cells. We performed target disruption of ataA by allelic marker exchange, and the resulting ΔataA strain was complemented with ataA on the Escherichia coli-Acinetobacter shuttle vector, which was newly constructed. These results proved that AtaA is essential for Tol 5’s autoagglutinating nature and high adhesiveness to surfaces of various materials. In addition, the adhesiveness to solid surfaces mediated by AtaA is notably higher than that mediated by YadA of Yersinia enterocolitica WA-314. Moreover, and importantly, these characteristics can be conferred to the non-adhesive, non-agglutinating bacterium Acinetobacter sp. ADP1 in trans by transformation with ataA, with expected applications to microbial immobilization.     

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.     

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.     

Monolayer adsorption of a "bald" mutant of the highly adhesive and hydrophobic bacterium Acinetobacter sp. strain Tol 5 to a hydrocarbon surface

The affinity of microbial cells for hydrophobic interfaces is important because it directly affects the efficiency of various bioprocesses, including green biotechnologies. The toluene-degrading bacterium Acinetobacter sp. strain Tol 5 has filamentous appendages and a hydrophobic cell surface, shows high adhesiveness to solid surfaces, and self-agglutinates. A "bald" mutant of this bacterium, strain T1, lacks the filamentous appendages and has decreased adhesiveness but retains a hydrophobic cell surface. We investigated the interaction between T1 cells and an organic solvent dispersed in an aqueous matrix. During a microbial-adhesion-to-hydrocarbon (MATH) test, which is frequently used to measure cell surface hydrophobicity, T1 cells adhered to hexadecane droplet surfaces in a monolayer, whereas wild-type cells aggregated on the droplet surfaces. The adsorbed T1 cells on the hexadecane surfaces hindered the coalescence of the droplets formed by vortexing, stabilizing the emulsion phase. Following the replacement of the aqueous phase with fresh pure water after the MATH test, a proportion of the T1 cells that had adsorbed to the hydrocarbon surface detached during further vortexing, suggesting a reversible adsorption of T1 cells. The final ratio of the adhering cells to the total cells in the detachment test coincided with that in the MATH test. The adhesion of T1 cells to the hydrocarbon surface conformed to the Langmuir adsorption isotherm, which describes reversible monolayer adsorption. Reversible monolayer adsorption should be useful for green technologies employing two-liquid-phase partitioning systems and for bioremediation because it allows effective reaction and transport of hydrophobic substrates at oil-water interfaces.     

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.     

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 was performed using the SoilMaster^™ DNA Extraction Kit (Epicentre, Madison, Wisconsin) and hybridization probe based real-time PCR. The detection target was the alkane hydroxylase gene (alkM). Standard curve construction showed a linear relation between log values of cell concentrations and real-time PCR threshold cycles over five orders of magnitude between 5.4±3.0×10⁶ and 5.4±3.0×10² CFU ml⁻¹ cell suspension. The detection limit was about 540 CFU ml⁻¹, which was ten times more sensitive than conventional PCR. The quantification of Acinetobacter sp. strain MUB1 cells in soil samples resulted in 46.67%, 82.41%, and 87.59% DNA recovery with a detection limit of 5.4±3.0×10⁴ CFU g⁻¹ dry soil. In this study, a method was developed for the specific, sensitive, and rapid quantification of the Acinetobacter sp. strain MUB1 in soil samples.     

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.     

Synergism of VAM and Rhizobium on Production and Metabolism of IAA in Roots and Root Nodules of Vigna Mungo

A novel Acinetobacter strain, Ud-4, possessing a strong capacity to degrade edible, lubricating, and heavy oil was isolated from seawater in a fishing port located in Toyama, Japan. It was identified by morphological and physiological analyses and 16S rDNA sequencing. This strain could utilize five types of edible oils (canola oil, olive oil, sesame oil, soybean oil, and lard), lubricating oil, and C-heavy oil as the sole carbon source for growth in M9 medium. The strain grew well and heavily degraded edible oils in Luria–Bertani medium during a 7-day culture at 25°C; it also degraded all kinds of oils in artificial seawater medium for marine bacteria. Furthermore, this strain was capable of degrading almost all C10–C25 n-alkanes in C-heavy oil during a 4-week culture. Oligonucleotide primers specific to two catabolic genes involved in the degradation of n-alkanes (Acinetobacter sp. alkM) and triglyceride (Acinetobacter sp. lipA) allowed amplification of these genes in strain Ud-4. To our knowledge, this is the first report on the isolation of a bacterium that can efficiently degrade both edible and mineral oils.     

Muricauda ruestringensis Has an Asymmetric Cell Cycle

Muricauda ruestringensis B1 is a Gram ‐negative, marine bacterium and a member of the Flavobacteriaceae family. It is characterized by long appendages, which appear at different stages of growth. At the outer end of these appendages there is a bulbous structure. Investigating the cell morphology of strain B1 during batch growth revealed a high diversity of cell types and sizes. Apart from small rod‐shaped cells and rods with appendages, there were large rods and spherical cells of different sizes as well as spherical cells which had fimbriae. To be able to study the cell cycle events, it was essential to monitor the population dynamics of the involved individuals. For this purpose, fluorochromising techniques, multi‐parametric flow cytometry, image analysis and fluorescence microscopy were used. It was demonstrated that all cell types displayed a broad variation in DNA content; the precise number of chromosomes varied depending on the growth phase. The assortment was testified to hold 16S rDNA sequence identity. The cultures consisted of subpopulations whose density within a Percoll gradient varied considerably, ranging from 1.028 to 1.070. Consolidating the results of the morphological data, the chromosome content and the density of the subpopulations at different growth stages enabled us to construct an asymmetric cell cycle for the growth of strain B1 under the specific culture conditions of our experiments.     

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.     

Enhanced Synthesis of the Exopolysaccharide Ethapolan by Acinetobacter sp. 12S Grown on a Mixture of Substrates

Enhanced synthesis of the exopolysaccharide ethapolan by Acinetobacter sp. 12S was observed when the bacterium was grown on a mixture of two energetically nonequivalent substrates (ethanol and glucose) taken in a molar proportion of 3.1 : 1. The efficiency of carbon transformation into EPSs was maximum when sodium ions were absent in the medium, the concentration of nitrogen source was reduced to 0.3–0.45 g/l, and the inoculum was grown on ethanol. Such conditions provided an increase in the maximum specific growth rate and its attainment in earlier cultivation terms. Molasses as a substitution for glucose was inefficient. The activities of the key enzymes of C₂ metabolism in Acinetobacter sp. 12S cells grown on the substrate mixture were 1.1 to 1.7 times lower than they were during growth on ethanol alone. The activity of isocitrate lyase in cells grown on the substrate mixture declined to an even greater extent (by 4–7 times), indicating that the role of the glyoxylate cycle in such cells is insignificant.     

Effect of dispersing oil phase on the biodegradability of a solid alkane dissolved in non-biodegradable oil

Acinetobacter sp. CR was grown on a model oil, which consisted of an inert oil matrix of pristane with n-heneicosane dissolved in it as the sole carbon source, in a stirred-tank bioreactor. This bacterium takes up substrates from the oil phase by direct contact with the oil phase. A previously established mathematical model was applied to reveal the effect of agitation conditions on the growth and n-alkane degradation kinetics of the bacterium. Higher impeller speed resulted in both lower microbial growth and lower n-alkane degradation rate of the bacterium, although it increased the specific surface area of the oil, which was measured by a previously developed device. This result was due to the decreased number of cells adhering to the oil surface, i.e., intense agitation inhibited the adhesion of cells to the oil surface. The addition of a surfactant below a critical micelle concentration (CMC) inhibited the degradation of n-heneicosane dissolved in pristane, although the biodegradability of the substrate recovered gradually with the increase in the dose of surfactant over CMC. The results suggest that efforts to increase the specific surface area of the oil phase have the undesirable result of inhibiting oil degradation when the dominant microbial degraders take up substrates in oil by direct contact with the oil. Electronic Publication     

Isolation and characterization ofAcinetobacter sp. mutants defective in exopolysaccharide biosynthesis

Nitrosoguanidine-induced mutants ofAcinetobacter sp. defective in exopolysaccharide biosynthesis did not differ from the parent strain in distinguishing physiological and biochemical properties, such as requirements for growth factors, utilization of mono- and disaccharides, and resistance to antibiotics. The genetic relation of parent and mutant strains was shown by 16S rRNA PCR analysis. The comparative study of parent and mutant strains with respect to resistance to unfavorable environmental factors confirmed our hypothesis thatAcinetobacter sp. exopolysaccharides perform protective functions. Hybridization experiments revealed the conjugal transfer of plasmid R68.45 fromPseudomonas putida BS228 (R68.45) to mutant but not to the parentAcinetobacter sp. strains. The role of theAcinetobacter sp. exopolysaccharides in providing the genetic stability of this bacterium is discussed.     

Antifouling properties of zinc oxide nanorod coatings

In laboratory experiments, the antifouling (AF) properties of zinc oxide (ZnO) nanorod coatings were investigated using the marine bacterium Acinetobacter sp. AZ4C, larvae of the bryozoan Bugula neritina and the microalga Tetraselmis sp. ZnO nanorod coatings were fabricated on microscope glass substrata by a simple hydrothermal technique using two different molar concentrations (5 and 10?mM) of zinc precursors. These coatings were tested for 5?h under artificial sunlight (1060?W?m^?2 or 530?W?m^?2) and in the dark (no irradiation). In the presence of light, both the ZnO nanorod coatings significantly reduced the density of Acinetobacter sp. AZ4C and Tetraselmis sp. in comparison to the control (microscope glass substratum without a ZnO coating). High mortality and low settlement of B. neritina larvae was observed on ZnO nanorod coatings subjected to light irradiation. In darkness, neither mortality nor enhanced settlement of larvae was observed. Larvae of B. neritina were not affected by Zn²⁺ ions. The AF effect of the ZnO nanorod coatings was thus attributed to the reactive oxygen species (ROS) produced by photocatalysis. It was concluded that ZnO nanorod coatings effectively prevented marine micro and macrofouling in static conditions.     

Intergeneric protoplast fusion between Acinetobacter sp. A3 and Pseudomonas putida DP99 for enchanced hydrocarbon degradation

Summary Polyethylene glycol 6000 mediated protoplast fusion between an alkane degrader Acinetobacter sp. A3, and a naphthalene degrader, Pseudomonas putida DP99 , resulted in fusants capable of degrading both hydrocarbons and were morphologically similar to Acinetobacter sp. A3. While fusant F4/13 and Pseudomonas putida DP99 degraded over 98% of naphthalene provided by the end of five days, tetradecane degradation by fusant F4/13 was 82% compared to 77% by Acinetobacter sp. A3 in the same time period. Also, while from naphthalene +tetradecane mixture, fusant F4/13 could degrade 99% and 53% of naphthalene and tetradecane respectively, both the parent strains together could degrade over 99% naphthalene but only about 16% tetradecane.     

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.     

Peculiarities of Ethanol Metabolism in an Acinetobacter sp. Mutant Strain Defective in Exopolysaccharide Synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD⁺-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD⁺ and NADP⁺ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C₄-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C₂-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

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.     

Isolation and characterization of sulfonamide-degrading bacteria Escherichia sp. HS21 and Acinetobacter sp. HS51

With the intensive application of sulfonamides in aquaculture and animal husbandry and the increase of sulfonamides discharged into the environments, there is an increasing need to find a way to remediate sulfonamide-contaminated environments. Two bacterial strains capable of degrading sulfonamides, HS21 and HS51, were isolated from marine environments. HS21 and HS51 were identified as members of Escherichia sp. and Acinetobacter sp., respectively, based on 16S rRNA gene sequencing. Degradation of each sulfonamide by Escherichia sp. HS21 and Acinetobacter sp. HS51 was characterized using capillary electrophoresis. About 66 or 72% of sulfapyridine and 45 or 67% of sulfathiazole contained in the media was degraded by Escherichia sp. HS21 or Acinetobacter sp. HS51, respectively, after incubation for 2 days. The supernatant from culture of Escherichia sp. HS21 or Acinetobacter sp. HS51 grown in sulfapyridine or sulfathiazole contained media had much attenuated cytotoxicity against HeLa cells. These results suggest that Escherichia sp. HS21 and Acinetobacter sp. HS51 are new bacterial resources for biodegrading sulfonamides and indicate the potential of isolated strains for the bioremediation of sulfonamide-polluted environments.