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.     

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.     

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.     

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.     

Induction of carbon monoxide dehydrogenase during heterotrophic growth of Acinetobacter sp. strain JC1 DSM 3803 in the presence of carbon monoxide

Abstract Experiments with crude cell extracts of Acinetobacter sp. JC1 revealed that CO dehydrogenase in this bacterium is inducible not only during chemoautotrophic growth with CO but also during heterotrophic growth in the presence of CO. The results indicate that Acinetobacter sp. JC1 can grow mixotrophically with organic material and CO.     

Adhesion of an Amylolytic Arthrobacter sp. to Starch-Containing Plastic Films

Cells of the amylolytic bacterium KB-1 (thought to be an Arthrobacter sp.) adhered (~70%) to the surface of plastic films composed of starch-poly (methylacrylate) graft copolymer (starch-PMA), but did not adhere (<10%) to films composed of polymethylacrylate (PMA), polyethylene (PE), carboxymethyl cellulose, or a mixture of PE plus poly (ethylene-coacrylic acid) (EAA), starch plus PE, or starch plus PE and EAA. About 30% of the cells adhered to gelatinized insoluble starch. Dithiothreitol (5 mM), EDTA (5 mM), and soluble starch (1%, wt/vol) had little effect on the adhesion of KB-1 cells to starch-PMA films. However, glutaraldehyde-fixed cells, azide-treated cells, and heat-killed cells did not bind to starch-PMA plastic, suggesting that the observed adhesion required cell viability. Culture supernatant from 5-day-old KB-1 cultures contained a proteolytic enzyme that inhibited cell adhesion to starch-PMA plastics. Trypsin-treated KB-1 cells also lost their ability to bind to starch-PMA plastic. When washed free of trypsin and suspended in fresh medium, trypsin-treated bacteria were able to recover adhesion activity in the absence, but not in the presence, of the protein synthesis inhibitor chloramphenicol. These results suggested that adhesion of KB-1 to starch-PMA plastic may be mediated by a cell surface protein. Although KB-1 bacteria bound to starch-PMA plastic, they did not appear to degrade starch in these films. Evidence of starch degradation was observed for starch-PE-EAA plastics, where <10% of the bacteria was bound, suggesting that cell adhesion may not be a prerequisite for degradation of some starch-containing plastics.     

Surface Physicochemistry and Ionic Strength Affects eDNA’s Role in Bacterial Adhesion to Abiotic Surfaces

Extracellular DNA (eDNA) is an important structural component of biofilms formed by many bacteria, but few reports have focused on its role in initial cell adhesion. The aim of this study was to investigate the role of eDNA in bacterial adhesion to abiotic surfaces, and determine to which extent eDNA-mediated adhesion depends on the physicochemical properties of the surface and surrounding liquid. We investigated eDNA alteration of cell surface hydrophobicity and zeta potential, and subsequently quantified the effect of eDNA on the adhesion of Staphylococcus xylosus to glass surfaces functionalised with different chemistries resulting in variable hydrophobicity and charge. Cell adhesion experiments were carried out at three different ionic strengths. Removal of eDNA from S. xylosus cells by DNase treatment did not alter the zeta potential, but rendered the cells more hydrophilic. DNase treatment impaired adhesion of cells to glass surfaces, but the adhesive properties of S. xylosus were regained within 30 minutes if DNase was not continuously present, implying a continuous release of eDNA in the culture. Removal of eDNA lowered the adhesion of S. xylosus to all surfaces chemistries tested, but not at all ionic strengths. No effect was seen on glass surfaces and carboxyl-functionalised surfaces at high ionic strength, and a reverse effect occurred on amine-functionalised surfaces at low ionic strength. However, eDNA promoted adhesion of cells to hydrophobic surfaces irrespective of the ionic strength. The adhesive properties of eDNA in mediating initial adhesion of S. xylosus is thus highly versatile, but also dependent on the physicochemical properties of the surface and ionic strength of the surrounding medium.     

Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress

The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM) and electrokinetics (electrophoresis). Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus). From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO(3), cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (～700-900 kPa and ～100-300 kPa respectively). Under similar ionic strength condition, a dramatic ～50% to ～70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.     

Acinetobacter sp. HM746599 isolated from leatherback turtle blood

A newly described bacterial isolate, Acinetobacter sp. HM746599, has been obtained from leatherback sea turtle hatchling blood. The implication is that the hatchling was infected during development in the egg, which is substantiated by other studies to be reported by us in the future. The 16S rRNA gene sequence of the bacterium (GenBank accession number: HM746599) showed the greatest similarity to the identified species, Acinetobacter beijerinckii (97.6-99.78%) and Acinetobacter venetianus (99.78%). Acinetobacter sp. HM746599 are gram-negative, rod-shaped coccobacilli and are hemolytic/cytotoxic to human and sea turtle red blood cells (RBCs). Hemolysis is not the result of any detectable soluble toxin. Acinetobacter beijerinckii and A. venetianus hemolyze sheep RBCs while Acinetobacter sp. HM746599 does not, and unlike A. venetianus, the growth of Acinetobacter sp. HM746599 and A. beijerinckii is not supported by l-arginine. Many Acinetobacter species, especially hemolytic ones, are pathogenic to immunologically compromised humans and it is possible that, in addition to sea turtles, this bacterium might also be a danger to susceptible humans who handle infected hatchlings. The bacteria are available from CCUG (Culture Collection, University Gothenburg, G?teborg, Sweden) and from NRRL (Agricultural Research Service Culture Collection, Peoria, IL).     

Adhesion of a <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain isolated from a paper machine to cellulose fibres

The adhesion to cellulose fibres of a strain of Pseudomonas putida isolated from a paper machine was studied under different environmental conditions. The physicochemical properties of both P. putida cells and cellulose fibres were also determined to better understand the adhesion phenomenon. Adhesion was rapid (1 min) and increased with time, cell concentration and temperature (from 25 to 40°C), indicating that bacterial adhesion to cellulose fibres is essentially governed by a physicochemical process. The P. putida cell surface was negatively charged, as shown by electrophoretic mobility measurements, and was hydrophilic due to a strong electron-donor character, as shown by the microbial adhesion to solvents method. Cellulose fibres were shown to be hydrophilic by contact angle measurements using the capillary rise method. These results suggest the importance of Lewis acid-base interactions in the adhesion process. In various ionic solutions (NaCl, KCl, CaCl₂ and MgCl₂), adhesion increased with increasing ionic strength up to 10–100 mM, indicating that, at low ionic strength, electrostatic interactions were involved in the adhesion process. An increase in the C/N ratio of the growth medium (from 5 to 90) decreased adhesion but this could not be related to changes in physicochemical properties, suggesting that other factors may be involved. In practice, temperature, ionic strength and nitrogen concentration must be taken into consideration to reduce bacterial contamination in the paper industry.     

Opportunistic colonization of Ralstonia solanacearum-infected plants by Acinetobacter sp. and its natural competence development

The behavior of the soil bacterium Acinetobacter sp. BD413 was monitored in Ralstonia solanacearum-infected and non-infected tomato plants after direct injection into the stem or natural infection by roots. In healthy plants, Acinetobacter sp. BD413 failed to colonize plant tissue. In plants infected simultaneously by the pathogen R. solanacearum,the Acinetobacter population increased linearly to about 3.1 x 10(7) cells per gram plant material and was maintained at a high level until the death of the plant. Moreover, Acinetobacter sp. BD413 was found to develop a competent state when multiplying in planta, indicating it could possibly be transformed by bacterial or plant DNA.     

低温菌启动子探针质粒的构建

为了在宿主菌Acinetobacter sp.DWC6中构建低温菌蛋白表达载体,以pBR322质粒为基础,去除质粒上β-内酰胺酶基因的启动子片段,取而代之为来源于质粒pJRD215的卡那霉素抗性基因片段,并在pBR322中插入Acinetobacter菌属特异性ori的DNA片段,构建了能在Acinetobacter sp.DWC6和E.coli中正常复制的启动子探针质粒pBAP1。通过在质粒pBAP1中的β-内酰胺酶基因上游随机导入Acinetobacter sp.DWC6基因组片段,通过检测宿主细胞的氨苄青霉素抗性和β-内酰胺酶活性,来筛选强启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。     

Activated T lymphocytes and macrophages secrete fibronectin which strongly supports cell adhesion.

Matrix-bound fibronectin (FN) appears to be involved in cell adhesion and motility mediated by integrin receptors. Although lymphoid cells and other cell types are capable of producing and secreting FN, the precise role of this secreted FN-like factor in regulating immune reactions is unclear. In the present study we analyzed the adhesive properties of FN secreted by rat CD4+ T cells and clone cells activated by the T cell mitogen concanavalin A (Con A), antigen, or via the CD2 pathways, or by macrophages (M phi) activated by lipopolysaccharide (LPS). Immobilized culture supernatant (CS) from the activated T cells or M phi supports the adhesion of activated rat or human CD4+ T cell or murine tumor cell. These CS contained FN and were more potent at facilitating cell adhesion then plasma FN. The adhesion activity of CS was attributed to FN because (a) gelatin columns depleted the FN present in the CS and (b) pretreating the cells with peptides of the cell-binding domain of FN abrogated their ability to bind CS. CS-mediated adhesion appears to occur primarily via the recognition of the Arg-Gly-Asp (RGD) by the beta 1-integrin-specific receptors of the adhesive cells. Thus, we postulate that FN secreted by various types of leukocytes is involved in promoting essential cell-matrix interactions, possibly affecting cell-adhesive and migratory processes at inflammatory or extravasation sites.     

T cell receptor-dependent, antigen-specific stimulation of a murine T cell clone induces a transient, VLA protein-mediated binding to extracellular matrix

Upon Ag stimulation, an arsonate-specific murine T cell clone exhibited a rapid but transient increase in cell adhesion to collagen, fibronectin, and laminin. This increase in cell adhesion was not observed when a mutant T cell clone lacking TCR expression was utilized. However, upon stimulation by phorbol esters, both parent and mutant T cell clones exhibited a similar transient increase in adhesion to the three matrix proteins. The observed cell adhesion was extensively inhibited by antibodies to the integrin beta 1 subunit, indicating the involvement of VLA proteins. Despite changes in the adhesive properties, there was essentially no difference in the expression of VLA-1, -3, -4, -5, and -6 between resting and stimulated T cells. Together these results suggest that Ag stimulation transmits signals via the TCR complex resulting in a rapid, but transient, up-regulation of matrix protein binding by VLA proteins already present at the cell surface. Because the appropriate reagents that recognize individual mouse VLA proteins were not available, we used the human T cell line Jurkat to demonstrate that T cell binding to collagen, laminin, and fibronectin is mediated largely by VLA-2, VLA-6, and a combination of VLA-5 and VLA-4, respectively.     

Adhesion of the positively charged bacterium Stenotrophomonas (Xanthomonas) maltophilia 70401 to glass and Teflon.

Medical implants are often colonized by bacteria which may cause severe infections. The initial step in the colonization, the adhesion of bacteria to the artificial solid surface, is governed mainly by long-range van der Waals and electrostatic interactions between the solid surface and the bacterial cell. While van der Waals forces are generally attractive, the usually negative charge of bacteria and solid surfaces leads to electrostatic repulsion. We report here on the adhesion of a clinical isolate, Stenotrophomonas maltophilia 70401, which is, at physiological pH, positively charged. S. maltophilia has an electrophoretic mobility of +0.3 x 10(-8) m2 V-1 s-1 at pH 7 and an overall surface isoelectric point at pH 11. The positive charge probably originates from proteins located in the outer membrane. For this bacterium, both long-range forces involved in adhesion are attractive. Consequently, adhesion of S. maltophilia to negatively charged surfaces such as glass and Teflon is much favored compared with the negatively charged bacterium Pseudomonas putida mt2. While adhesion of negatively charged bacteria is impeded in media of low ionic strength because of a thick negatively charged diffuse layer, adhesion of S. maltophilia was particularly favored in dilute medium. The adhesion efficiencies of S. maltophilia at various ionic strengths could be explained in terms of calculated long-range interaction energies between S. maltophilia and glass or Teflon.     

Alcohol inhibits cell-cell adhesion mediated by human L1 [published erratum appears in J Cell Biol 1996 Jun;133(5):1139-40]

Mental retardation, hydrocephalus, and agenesis of the corpus callosum are observed both in fetal alcohol syndrome (FAS) and in children with mutations in the gene for the cell adhesion molecule L1. We studied the effects of ethanol on cell-cell adhesion in mouse fibroblasts transfected with human L1. L1-transfected fibroblasts exhibited increased cell-cell adhesion compared with wild-type or vector- transfected controls. Ethanol potently and completely inhibited L1- mediated adhesion both in transfected L cells and NIH/3T3 cells. Half- maximal inhibition was observed at 7 mM ethanol, a concentration achieved in blood and brain after ingesting one alcoholic beverage. In contrast, ethanol did not inhibit the adhesion of fibroblasts transfected with vector alone or with N-CAM-140. L1-mediated cell-cell adhesion was inhibited with increasing potency by n-propanol and n- butanol, but was not inhibited at all by n-alcohols of 5 to 8 carbons, acetaldehyde, or acetate, suggesting that ethanol interacts directly with a small hydrophobic pocket within L1. Phenylalanine, teratogenic anticonvulsants, and high concentrations of glucose did not inhibit L1- mediated cell-cell adhesion. Ethanol also inhibited potently the heterotypic adhesion of rat cerebellar granule cells to a monolayer of L1-transfected NIH/3T3 cells, but had no effect on their adhesion to N- CAM-140 or vector-transfected NIH/3T3 cells. Because L1 plays a role in both neural development and learning, ethanol inhibition of L1-mediated cell-cell interactions could contribute to FAS and ethanol-associated memory disorders.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

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. DeltaastA(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.