Utilization of surface localized substrate by non-adhesive marine bacteria

Thirty-four marine bacteria were isolated from the eluate of seawater passed through a column of glass beads coated with stearic acid. Irreversible attachment of these isolates to stearic acid-coated glass surfaces ranged from 7.6–100% of the total attached population, with 7 isolates exhibiting less than 10% irreversible adhesion. All 14 isolates tested were able to utilize surface bound¹⁴C-stearic acid, even though some showed mostly reversible adhesion to the surface. More detailed studies were made comparing the reversibly adheringVibrio MH3 with the irreversibly adheringPseudomonas NCMB2021. MH3 cells were readily removed from the surface by a gentle shear force, and a significant degree of¹⁴C-labeling of MH3 cells, but not of NCMB2021 cells, in the bulk phase was observed. The ecological significance of nutrient scavenging at solid surfaces by reversibly attached bacteria is considered.     

Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis.

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Behavior ofPseudomonas fluorescens within the hydrodynamic boundary layers of surface microenvironments

Phase, darkfield, and computer-enhanced microscopy were used to observe the surface microenvironment of flow cells during bacterial colonization. Microbial behavior was consistent with the assumptions used previously to derive surface colonization kinetics and to calculate surface growth and attachment rates from cell number and distribution. Surface microcolonies consisted of closely packed cells. Each colony contained 2ⁿ cells, where n is the number of cell divisions following attachment. Initially, cells were freely motile while attached, performing circular looping movements within the plane of the solid-liquid interface. Subsequently, cells attached apically, maintained a fixed position on the surface, and rotated. This type of attachment was reversible and did not necessarily lead to the formation of microcolonies. Cells became irreversibly attached by progressing from apical to longitudinal attachment. Longitudinally attached cells increased in length, then divided, separated, moved apart laterally, and slid next to one another. This resulted in tight cell packing and permitted simultaneous growth and adherence. After approximately 4 generations, individual cells emigrated from developing microcolonies to recolonize the surface at new locations. Surface colonization byPseudomonas fluorescens can thus be subdivided into the following sequential colonization phases: motile attachment phase, reversible attachment phase, irreversible attachment phase, growth phase, and recolonization phase.     

Binding and cytotoxicity of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and erythrocytes

The binding of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and human erythrocytes was studied in detail. Scatchard plots of binding of 125I-lectins to these cells gave biphasic lines except for HeLa cells at 0 degree C. The association constants of lectins for the three cell types at 37 degrees C were lower than those at 0 degree C. The numbers of total binding sites were estimated to be 7 to 16 X 10(7) per HeLa cell, 3 to 4 X 10(7) per Sarcoma 180 ascites tumor cell and 0.4 to 1 X 10(6) per erythrocyte. A fraction, 16 to 27% of the total amount of cell-bound lectin at 37 degrees C, appeared to be bound irreversibly as judged by non-removal on washing with 0.1 M lactose, whereas no lectin was irreversibly bound at 0 degree C. In the case of erythrocytes, no lectin became irreversibly bound even at 37 degrees C. The toxicity of lectins on HeLa cells and Sarcoma 180 ascites tumor cells was investigated. The toxicity of ricin D was 50 times for Sarcoma 180 ascites tumor cells and 140 times for HeLa cells as much as that for castor bean hemagglutinin. As to the sensitivities of both cell types to these lectins, it became apparent that Sarcoma 180 ascites tumor cells were more susceptible than HeLa cells.     

The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

Firm but slippery attachment of Deinococcus geothermalis

Bacterial biofilms impair the operation of many industrial processes. Deinococcus geothermalis is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria. It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known. High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of D. geothermalis E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium. Extensive slime matrix was absent from the D. geothermalis E50051 biofilms. AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed. We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible. In spite of this, AFM imaging of living D. geothermalis E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells. The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface. Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water. Biofilms were evaluated for their strength of attachment. D. geothermalis E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of Burkholderia cepacia F28L1 or the well-characterized biofilm former Staphylococcus epidermidis O-47. Deinococcus radiodurans strain DSM 20539(T) also formed tenacious biofilms. This paper shows that D. geothermalis has firm but laterally slippery attachment not reported before for a nonmotile species.     

Antibody inhibition of HeLa cell invasion by Yersinia enterocolitica

Antisera were prepared in rabbits against formalized and heat-killed bacteria of Yersinia enterocolitica serotype O:5,27 and against formalized bacteria of serotype O:8. Both strains used for immunization demonstrated adhesion to and invasion of HeLa cells. Coating of the bacteria with antibody did not greatly alter adhesion (i.e., extracellular attachment) to HeLa cells; however, antibody against formalized bacteria of both serotypes inhibited HeLa cell invasion by the homologous and heterologous strains. The Fab fragments from purified immunoglobulins also demonstrated cross-reacting inhibition of HeLa cell invasion. Antibody against heat-killed bacteria of serotype O:5,27 had no inhibitory activity. Adsorption of the antiserum against formalized bacteria of serotype O:5,27 with lipopolysaccharide from the homologous strain removed anti-lipopolysaccharide antibody but did not remove the inhibitory activity. The antiserum against formalized bacteria of serotype O:8 showed no antibody against lipopolysaccharide from serotype O:5,27 and no agglutinins against heat-killed bacteria of this strain. From these results, it is tentatively suggested that protein structures are important in mediating epithelial cell invasion by Y. enterocolitica.     

Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.

Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.     

A requirement for reversible binding between aggregating embryonic cells before stable adhesion.

Chick embryonic liver and neural retina cells aggregate in a two-step process. Initially, cells formed a loose association in a step that apparently did not require metabolic energy. Cells bound in this manner were dissociable by mild shear forces or by simple dilution. The results of the dilution experiments suggest a readily reversible binding of single cells to form these types of aggregates. In a second step, which required metabolic energy, the cells became firmly, or stably attached. The formation of both types of bond was temperature-dependent. Kinetic studies indicated that the formation of reversible bonds between cells was required before the cells could become stably attached, and that reversibly bound cells were converted directly into stably bound cells.     

Reversible and irreversible adhesion of motile Escherichia coli cells analyzed by total internal reflection aqueous fluorescence microscopy

The initial events in bacterial adhesion are often explained as resulting from electrostatic and van der Waals forces between the cell and the surface, as described by DLVO theory (developed by Derjaguin, Landau, Verwey, and Overbeek). Such a theory predicts that negatively charged bacteria will experience greater attraction toward a negatively charged surface as the ionic strength of the medium is increased. In the present study we observed both smooth-swimming and nonmotile Escherichia coli bacteria close to plain, positively, and hydrophobically coated quartz surfaces in high- and low-ionic-strength media by using total internal reflection aqueous fluorescence microscopy. We found that reversibly adhering cells (cells which continue to swim along the surface for extended periods) are too distant from the surface for this behavior to be explained by DLVO-type forces. However, cells which had become immobilized on the surface did seem to be affected by electrostatic interactions. We propose that the "force" holding swimming cells near the surface is actually the result of a hydrodynamic effect, causing the cells to swim at an angle along the glass, and that DLVO-type forces are responsible only for the observed immobilization of irreversibly adhering cells. We explain our observations within the context of a conceptual model in which bacteria that are interacting with the surface may be thought of as occupying one of three compartments: bulk fluid, near-surface bulk, and near-surface constrained. A cell in these compartments feels either no effect of the surface, only the hydrodynamic effect of the surface, or both the hydrodynamic and the physicochemical effects of the surface, respectively.     

Reversible and Irreversible Adhesion of Motile Escherichia coli Cells Analyzed by Total Internal Reflection Aqueous Fluorescence Microscopy

The initial events in bacterial adhesion are often explained as resulting from electrostatic and van der Waals forces between the cell and the surface, as described by DLVO theory (developed by Derjaguin, Landau, Verwey, and Overbeek). Such a theory predicts that negatively charged bacteria will experience greater attraction toward a negatively charged surface as the ionic strength of the medium is increased. In the present study we observed both smooth-swimming and nonmotile Escherichia coli bacteria close to plain, positively, and hydrophobically coated quartz surfaces in high- and low-ionic-strength media by using total internal reflection aqueous fluorescence microscopy. We found that reversibly adhering cells (cells which continue to swim along the surface for extended periods) are too distant from the surface for this behavior to be explained by DLVO-type forces. However, cells which had become immobilized on the surface did seem to be affected by electrostatic interactions. We propose that the “force” holding swimming cells near the surface is actually the result of a hydrodynamic effect, causing the cells to swim at an angle along the glass, and that DLVO-type forces are responsible only for the observed immobilization of irreversibly adhering cells. We explain our observations within the context of a conceptual model in which bacteria that are interacting with the surface may be thought of as occupying one of three compartments: bulk fluid, near-surface bulk, and near-surface constrained. A cell in these compartments feels either no effect of the surface, only the hydrodynamic effect of the surface, or both the hydrodynamic and the physicochemical effects of the surface, respectively.     

Surface contact stimulates the just-in-time deployment of bacterial adhesins

The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive.     

Effects of acid pH and urea on the spectral properties of the LHII antenna complex from the photosynthetic bacterium Ectothiorhodospira sp.

The aim of this study was to investigate the spectral modifications of the LHII antenna complex from the purple bacterium Ectothiorhodospira sp. upon acid pH titration both in the presence and absence of urea. A blue shift specifically and reversibly affected the B850 band around pH 5.5-6.0 suggesting that a histidine residue most probably participated in the in vivo absorption red shifting mechanism. This transition was observed in the presence and absence of urea. Under strong chaotropic conditions, a second transition occurred around pH 2.0, affecting the B800 band irreversibly and the B850 reversibly. Under these conditions a blue shift from 856 to 842 nm occurred and a new and strong circular dichroism signal from the new 842 nm band was observed. Reverting to the original experimental conditions induced a red shift of the B850 band up to 856 nm but the circular dichroism signal remained mostly unaffected. Under the same experimental conditions, i.e. pH 2.1 in the presence of urea, part of the B800 band was irreversibly destroyed with concomitant appearance of a band around 770 nm due to monomeric bacteriochlorophyll from the disrupted B800. Furthermore, Gaussian deconvolution and second derivative of the reverted spectra at pH 8.0 after strong-acid treatment indicated that the new B850 band was actually composed of two bands centered at 843 and 858 nm. We ascribed the 858 nm band to bacteriochlorophylls that underwent reversible spectral shift and the 843 nm band to oligomeric bacteriopheophytin formed from a part of the B850 bacteriochlorophyll. This new oligomer would be responsible for the observed strong and mostly conservative circular dichroism signal. The presence of bacteriopheophytin in the reverted samples was definitively demonstrated by HPLC pigment analysis. The pheophytinization process progressed as the pH decreased below 2.1, and at a certain point (i.e. pH 1.5) all bacteriochlorophylls, including those from the B800 band, became converted to oligomeric bacteriopheophytin, as shown by the presence of a single absorption band around 843 nm and by the appearance of a single main elution peak in the HPLC chromatogram which corresponded to bacteriopheophytin.     

Competitive adherence as a mechanism of bacterial interference

To determine whether competition among bacteria for specific attachment sites on host cells can explain bacterial interference, Staphylococcus aureus strain 502A was tested in turn against two different nasal coryneforms, a strain of Pseudomonas aeruginosa, and a virulent strain of S. aureus, all in the presence of nasal mucosal cells. Particularly examined was the influence of sequence in which bacteria were presented to the nasal cells in comparison with initial mixtures and individual suspensions. Results paralleled those observed in clinical prophylaxis: the bacterium first to adhere to the epithelial cells was able, under uniform conditions, to interfere with the colonization of subsequently added bacteria. Secondary adherence was not eliminated but substantially reduced, and was probably related to steric blockage by the initial colonizer and its particular ability to dissociate from the host cell.     

Redox properties and active center of phototrophic bacteria hydrogenases

It is shown that the activity of phototrophic bacteria hydrogenases depends on the redox potential (Eh) of the medium. Hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina strain BBS reversibly activates H2 at Eh less than -290 mV (pH 7.0). When Eh is increased from -290 to -170 mV, the enzyme is converted into an inactive form which is accompanied by one-electron oxidation of its Fe-S cluster. In contrast, the hydrogenases of the purple nonsulfur bacterium Rhodobacter capsulatus B10 and the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum exhibit maximum activity at Eh greater than -300 mV, favourable only for H2 uptake. When Eh decreases the activities of these enzymes drop dramatically; this accounts for their unidirectional effect directed mainly towards H2 uptake. Such dependence on Eh of activity of hydrogenases from these bacteria correlates with their physiological function in the metabolism of phototrophic bacteria, i.e. with the catalysis of the H2 uptake reaction. Hydrogenases from purple bacteria contain nickel and a single Fe-S cluster. Metal chelators do not affect the activity of these enzymes, which indicates that iron and nickel are tightly bound to the apoprotein. Sulfhydryl compounds irreversibly inactivate T. roseopersicina hydrogenase by 30-40% in the presence of sulfide. Acetylene and carbon monoxide are reversible inhibitors of the enzyme. EPR and inhibitory analysis indicate a direct interaction of H2 with the nickel ion in the active center of the T. roseopersicina hydrogenase.     

Interaction of Bacteriophage N1 with Cell Walls of Micrococcus lysodeikticus

Bacteriophage N1 does not irreversibly adsorb to cell walls isolated from its host Micrococcus lysodeikticus strain 1 (ML-1). ML-1 walls do bind the virus in a specific but completely reversibly union. Electron microscopic examination of OsO(4)-treated mixtures of phage and walls revealed phage bound to wall fragments by their tail tips, suggesting that reversible phage attachment to walls involves a "tail-first" adsorption of the virus. Treatment of ML-1 walls with fluorodinitrobenzene confers upon the walls the ability to inactivate N1 phage. The relationship between reversible phage attachment to walls and the mechanism of infection by N1 phage is discussed.     

Evidence for the development of an intermonomeric asymmetry in the covalent binding of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to human erythrocyte band 3

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies have identified two oligomeric forms of band 3 whose proportions on gel profiles were modulated by the particular ligand occupying the intramonomeric stilbenedisulfonate site during intermonomeric cross-linking by BS3 [bis-(sulfosuccinimidyl) suberate] [Salhany et al. (1990) J. Biol. Chem. 265, 17688-17693]. When DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) was irreversibly attached to all monomers, BS3 covalent dimers predominated, while with DNDS (4,4'-dinitrostilbene-2,2'-disulfonate) present to protect the intramonomeric stilbenedisulfonate site from attack by BS3, a partially cross-linked band 3 tetramer was observed. In the present study, we investigate the structure of the protected stilbenedisulfonate site within the tetrameric complex by measuring the ability of patent monomers to react irreversibly with DIDS. Our results show two main populations of band 3 monomers present after reaction with DNDS/BS3: (a) inactive monomers resulting from the displacement of reversibly bound DNDS molecules and subsequent irreversible attachment of BS3 to the intramonomeric stilbenedisulfonate site and (b) residual, active monomers. All of the residual activity was fully inhibitable by DIDS under conditions of reversible binding, confirming expectations that all of the monomers responsible for the residual activity have patent stilbenedisulfonate sites. However, within this active population, two subpopulations could be identified: (1) monomers which were irreversibly reactive toward DIDS and (2) monomers which were refractory toward irreversible binding of DIDS at pH 6.9, despite being capable of binding DIDS reversibly. Increasing the pH to 9.5 during treatment of DNDS/BS3-modified cells with 300 microM DIDS did not cause increased irreversible transport inhibition relative to that seen for cells treated at pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of larval barnacle attachment to bacterial films: An investigation of physical properties

The effects of films of two strains of a marine bacterium, Deleya marina (ATCC 25374 and 27129) on the attachment response of cypris larvae of the balanomorph barnacle, Balanus amphitrite, were examined in the laboratory. Tests showed that the cell-surface hydrophobicities of the two bacteria in suspension were different. In contrast, films derived from these cells were both highly wettable (i.e., displayed high surface free energy). Assays (22 hours) compared permanent attachment of larval barnacles to films derived from exponential and stationary phase cells for both bacteria. These films either had no effect or inhibited attachment of both 0-day- and 4-day-old cypris larvae when compared with unfilmed controls. Our data indicate that inhibition of larval barnacle attachment by films of the two bacteria is the result of factors other than surface free energy. Production of chemical barnacle settlement inhibitors by the bacteria is hypothesized.Offprint requests to: J. S. Maki.