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A thermophilic syntrophic bacterium, Pelotomaculum thermopropionicum strain SI, was grown in a monoculture or coculture with a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ⌬H. Microscopic observation revealed that cells of each organism were dispersed in a monoculture independent of the growth substrate. In a coculture, however, these organisms coaggregated to different degrees depending on the substrate; namely, a large fraction of the cells coaggregated when they were grown on propionate, but relatively few cells coaggregated when they were grown on ethanol or 1-propanol. Field emission-scanning electron microscopy revealed that flagellum-like filaments of SI cells played a role in making contact with ⌬H cells. Microscopic observation of aggregates also showed that extracellular polymeric substance-like structures were present in intercellular spaces. In order to evaluate the importance of coaggregation for syntrophic propionate oxidation, allowable average distances between SI and ⌬H cells for accomplishing efficient interspecies hydrogen transfer were calculated by using Fick's diffusion law. The allowable distance for syntrophic propionate oxidation was estimated to be approximately 2 m, while the allowable distances for ethanol and propanol oxidation were 16 m and 32 m, respectively. Considering that the mean cell-to-cell distance in the randomly dispersed culture was approximately 30 m (at a concentration in the mid-exponential growth phase of the coculture of 5 ؋ 10 7 cells ml ؊1 ), it is obvious that close physical contact of these organisms by coaggregation is indispensable for efficient syntrophic propionate oxidation.In anaerobic digestors, organic matter is converted to methane and CO 2 via various intermediates (1, 27). Among the most important intermediate metabolites are volatile fatty acids (VFAs), such as acetate, propionate, and butyrate, and it has been reported that accumulation of VFAs results in a significant decrease in the methane production efficiency in such digestors (16,17,37,38). VFAs are, however, unfavorable substrates for anaerobes, since oxidation of these substrates to H 2 and CO 2 (or formate) is endoergonic under standard conditions (i.e., the changes in the Gibbs free energy are positive [⌬G°Ј Ͼ 0]) (Table 1) and is thermodynamically feasible only when the H 2 partial pressure (or formate concentration) is kept low (3,11,29,34). For instance, thermodynamic estimation has predicted that H 2 partial pressures as low as 10 Pa and 100 Pa are necessary for the oxidation of propionate and butyrate, respectively (29, 34). Since H 2 and formate are scavenged mainly by the carbonate respiration of methanogenic archaea, syntrophic association of VFA-oxidizing bacteria (called syntrophs) and methanogenic archaea is considered indispensable for efficient VFA oxidation (27, 36).As described previously, syntrophic VFA oxidation depends on interspecies electron (as H 2 and formate) transfer. Researchers have suggested that close physical contact between syntro...

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.

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 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.Microbial adhesion is detrimental to both human life and industrial processes, causing infection and contamination by pathogens, dental decay, and biofilm formation, but it can also be beneficial in some environmental bioprocesses and in agriculture (5). Therefore, microbial adhesion has attracted much attention from researchers in various fields (2,5,8,20). Exopolymeric substances (EPS) (10,11,14,17) and proteinaceous appendages such as pili (4, 21) and flagella (3, 9) have been shown to be responsible for tenacious bacterial adhesion by forming a bridge between a cell and a surface. We previously isolated a rod-shaped, gram-negative, toluene-degrading bacterium, strain Tol 5, which was classified as Acinetobacter sp. (genomospecies 10) by 16S rRNA sequence analysis and physiological tests using the Micro Station system (BIOLOG) (13). This strain has a highly hydrophobic cell surface and is highly adhesive to solid surfaces. We have found that this adhesive property is beneficial to a biofiltration process for treating off gas containing volatile organic chemicals such as toluene because it allows the effective immobilization and accumulation of these bacterial cells with their high degradation activity on carrier materials. In the current study, we describe the cell appendages that mediate the adhesion of this bacterium to a solid surface.Bacterial cell appendages and EPS have been visualized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Compared with SEM, TEM provides high-resolution images by operating at higher electron-accelerating voltages (usually 70 to 100 kV for biotic samples). Of the several TEM techniques that have been used to observe living specimens (7,11,16,17), negative staining methods produce images that reflect the intact morphology of cell surface structures; such structures are unaffected by sample preparation because, in this method, the sample is mounted on a grid and subjected to electron staining without any other pretreatment. Indeed, many researchers have used TEM coupled with negative staining to visualize bacterial cell surface appendages involved in cell adhesion (9,12,15,21).To observe the cell surface structure of Tol 5 by TEM, the cells were grown until stationary phase at 28°C in 20 ml of a basal salt medium (Na 2 HPO 4 , 4.9 g; KH 2 PO 4 , 2.0 g; (NH 4 .2]) supplemented with 10 l of toluene in a 100-ml Erlenmeyer flask (13). To the flask were added four pieces (1.15 by 1.15 by 1.00 cm) of sponge carrier made of polyurethane, wit...

Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.

Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three ␤-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner ␤-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions.

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