Surface Exclusion Research Articles

Measuring Partition Coefficients of Colloids at Air−Water Interfaces

Quantification of surface excesses of colloidal particles at gas-liquid interfaces under environmentally relevant conditions has not previously been reported, despite its importance in understanding and predicting partitioning, transport, and transformations of colloids and other organic and metal species complexed onto colloids in many natural environments and engineered systems. In this study, the authors developed a bubble column method for measuring partition coefficients of colloids at air-water interfaces. The method was validated by comparing the adsorption isotherm constant for a surfactant measured using the authors` method with that determined from conventional surface tension measurements and the Gibbs absorption equation. The first measured partition coefficients of kaolinite and humic colloids at the air-water interfaces are reported. The bubble column method can also be used to identify surface exclusion of colloids at the air-water interface. By extension, this method will permit quantification of surface activities of a wide range of inorganic, organic, and microbial colloids as well as molecular species complexed onto colloids.

Genetic analysis of the role of the transfer gene, traN, of the F and R100-1 plasmids in mating pair stabilization during conjugation.

Mating pair stabilization occurs during conjugative DNA transfer whereby the donor and recipient cells form a tight junction which requires pili as well as TraN and TraG in the donor cell. The role of the outer membrane protein, TraN, during conjugative transfer was examined by introduction of a chloramphenicol resistance cassette into the traN gene on an F plasmid derivative, pOX38, to produce pOX38N1::CAT. pOX38N1::CAT was greatly reduced in its ability to transfer DNA, indicating that TraN plays a greater role in conjugation than previously thought. F and R100-1 traN were capable of complementing pOX38N1::CAT transfer equally well when wild-type recipients were used. F traN, but not R100-1 traN, supported a much lower level of transfer when there was an ompA mutation or lipopolysaccharide (LPS) deficiency in the recipient cell, suggesting receptor specificity. The R100-1 traN gene was sequenced, and the gene product was found to exhibit 82.3% overall similarity with F TraN. The differences were mainly located within a central region of the proteins (amino acids 162 to 333 of F and 162 to 348 of R100-1). Deletion analysis of F traN suggested that this central portion might be responsible for the receptor specificity displayed by TraN. TraN was not responsible for TraT-dependent surface exclusion. Thus, TraN, and not the F pilus, appears to interact with OmpA and LPS moieties during conjugation, resulting in mating pair stabilization, the first step in efficient mobilization of DNA.

Absence of Surface Exclusion in the First Stage of Lysozyme Adsorption Is Driven through Electrostatic Self-Assembly

Adsorption kinetics of hen egg white lysozyme at pH 7.4 onto a Si(Ti)O2 surface were measured at two ionic strengths and under different shear rates by means of optical waveguide lightmode spectroscopy. At low ionic strength, increases of the adsorbed amount linear with time suggest that the adsorption proceeds without surface exclusion. Within the concentration range investigated, the duration of this linear regime was inversely proportional to the bulk concentration. The linear regime always stopped at a threshold value of 0.18 ± 0.02 μg/cm2 and was followed by a second regime with surface exclusion. At high ionic strength the linear regime was not observed, suggesting an electrostatically driven self-assembly process at low ionic strength. This conclusion is supported by the linear arrays of lysozyme at graphite surfaces observed with STM (Haggerty, L.; Lenhoff, A. M. Biophys. J. 1993, 64, 886). Moreover, the adsorption rate at low ionic strength is faster than that predicted by diffusion across the di...

Modeling colloid transport and deposition in saturated fractures

A model is developed to describe the transport of colloids in a saturated fracture with a spatially variable aperture, accounting for colloid deposition onto fracture surfaces under various physicochemical conditions. The fracture plane is partitioned into unit elements with different apertures generated stochastically from a log-normal distribution. The model also accounts for colloid size exclusion from fracture elements with small apertures. Both equilibrium and kinetic colloid deposition onto fracture surfaces are investigated. Colloid surface exclusion is incorporated in the dynamics of kinetic deposition. The impact of deposited colloids on further colloid deposition is described by either a linear or a non-linear blocking function. The resulting system of governing partial differential equations is solved numerically using the fully implicit finite difference method. Model simulations illustrate the presence of preferential colloid transport in the fracture plane. It is shown that size exclusion increases the dispersion of colloids and leads to earlier breakthrough, especially for large-size particles. Furthermore, it is demonstrated that surface exclusion enhances colloid transport, and the assumption of clean-bed media may underestimate liquid-phase colloid concentrations.

Fluctuations in the number of particles adsorbed under the influence of diffusion and flow

Fluctuations in the number of colloid particles adsorbed irreversibly under diffusion and flow were determined. The experimental measurements were carried out in the impinging-jet cells using as model colloids the monodisperse polystyrene latex particles of micrometer size range adsorbing at mica sheets. The surface concentration of adsorbed particles was determined quantitatively using the direct microscope observation method coupled with an image analyzing system. Two series of experiments were performed (i) for diffusion controlled adsorption when the random sequential adsorption (RSA) mechanism was valid and (ii) for flow controlled adsorption. It was found that in the case of RSA the reduced variance of the distributions decreased markedly for increasing surface concentration θ in accordance with theoretical predictions based on the mean-field approximation. The experimental results were in a good agreement with the numerical simulations performed according to the RSA algorithm. It was also determined that the magnitude of fluctuations in our irreversible system was very similar to reversible systems described by the scaled-particle theory. A significantly different behavior was observed for flow affected adsorption when the reduced variance (at the same surface concentration) was much smaller than for the RSA model, therefore deviating considerably from an equilibrium system. The decrease in the variance indicated that the surface exclusion effects (described by the available surface function) were more important under flow due to the hydrodynamic scattering effect.

Redundant homosexual F transfer facilitates selection-induced reversion of plasmid mutations

F plasmids use surface exclusion to prevent the redundant entry of additional F plasmids during active growth of the host cells. This mechanism is relaxed during stationary phase and nonlethal selections, allowing homosexual redundant plasmid transfer. Homosexual redundant transfer occurs in stationary-phase liquid cultures, within nongrowing populations on solid media, and on media lacking a carbon source. We examined the relationship between homosexual redundant transfer, which occurs between F+ hosts, and reversion of a plasmid-encoded lac mutant allele, lacI33omegalacZ. Sodium dodecyl sulfate (SDS) and mutations that prevent normal transfer to F- cells reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. A recA null mutation reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ mutation. Conversely, a recD null mutation increased redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. These results suggest an explanation for why SDS and these mutations affect reversion of the plasmid lacI33omegalacZ allele. However, a direct causal relationship between transfer and reversion remains to be established. These results suggest that Rec proteins play an active role in redundant transfer and/or that redundant transfer is regulated with the activation of recombination. Redundant homosexual plasmid transfer during a period of stress may represent a genetic response that facilitates evolution of plasmid-encoded functions through mutation, recombination, reassortment, and dissemination of genetic elements present in the populations.

Molecular cloning of determinants of surface exclusion of F-like plasmid pAP22-2

Molecular cloning of the SalI-fragment of plasmid pAP22-2 DNA was carried out in order to detect the possible location of surface exclusion genes of this plasmid (Sfx VII system). The results of studying recombinant plasmids indicate that Sfx domain determinants are located in two different fragments of plasmid pAP22-2.

Surface Area Exclusion Chromatography: Influence of Localized and Mobile Adsorption Processes

Surface exclusion chromatography is particularly suitable to determine the structure of highly dilute solutions, where more common methods such as light scattering are inefficient and other chromatographic techniques cannot be employed due to strong adsorption of the solutes on the stationary phases. This process was simulated using two kinetic models (localized and mobile adsorption) to randomly deposit solute particles on the adsorbent plates of a chromatographic column. The influence of the relative size and concentration of two different adsorbing species on the degree of coverage of the successive plates of the column was investigated, and chromatograms derived from simulation were compared to those for the chromatographic separation by mutual surface exclusion of unimers and micelles in solutions of a diblock copolymer.

Transcriptional analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

The prgB gene encodes aggregation substance (Asc10) which is essential for transfer of the pheromone-inducible conjugative plasmid pCF10 in Enterococcus faecalis. The prgQ and prgS regions, located 4 kb upstream of prgB, are required for the expression of prgB. Complementation studies indicated that the prgQ region functions in cis and in an orientation-dependent manner relative to the prgB gene (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). Analysis of transcriptional fusions in this study, using a promoterless lacZ gene in several locations between prgQ and prgB, confirmed that the prgQ region does not carry a promoter for the expression of prgB and that prgB does not comprise an operon with prgA (which encodes the surface exclusion protein Sec10), the gene immediately upstream from prgB. Northern (RNA) blot analysis demonstrated that two distinct transcripts (Qs RNA and QL RNA), much larger than the prgQ gene, were expressed in the prgQ region. QS RNA was produced constitutively, whereas QL RNA was produced inducibly by pheromone. The lack of any other open reading frame in QL RNA and significant sequence complementarity between the 3' end of QL RNA and the promoter region of prgB suggested that the functional products of the prgQ region might be RNA molecules rather than proteins. A mutation in prgS completely abolished the production of QL RNA. A model for transcriptional activation of prgB is presented.

Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions

The prgB gene encodes the surface protein Asc10, which mediates cell aggregation resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis. Previous Tn5 insertional mutagenesis and sequencing analysis of a 12-kb fragment of pCF10 indicated that a region containing prgX, -Q, -R, -S, and -T, located 3 to 6 kb upstream of prgB, is required to activate the expression of prgB. Complementation studies showed that the positive regulatory region functions in cis in an orientation-dependent manner (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). In order to determine the involvement of each gene in the activation of prgB, Tn5 insertional mutagenesis and exonuclease III deletion analyses of the regulatory region were carried out. The results indicate that prgQ and -S are required for the expression of prgB, while prgX, -R, and -T are not required. Western blot (immunoblot) analysis of these mutants shows that prgQ is also essential for the expression of prgA (encoding the surface exclusion protein Sec10), which is located between prgB and the positive-control region. Complementation analysis demonstrates that a cis-acting regulatory element is located in the prgQ region and that pCF10 sequences in an untranslated region 3' from prgQ are an essential component of the positive-control system. Analyses of various Tn5 insertions in pCF10 genes suggest that transcription reading into this transposon is terminated in E. faecalis but that outward-reading transcripts may initiate from within the ends of Tn5 or from the junction sequences.

Redundant transfer of F' plasmids occurs between Escherichia coli cells during nonlethal selections.

Surface exclusion is the mechanism by which F plasmids prevent the redundant entry of additional F plasmids into the host cell during exponential growth. This mechanism is relaxed in cells that are in stationary phase. Using genetically marked F' plasmids and host strains, we extend this finding to Escherichia coli populations during extended nonlethal selection in bacterial lawns. We show that a high level of redundant transfer occurs between these nongrowing cells during the selection. This result has implications for the mechanism of adaptive mutagenesis.

The Surface Exclusion System of RP1: Investigation of the Roles of trbJ and trbK in the Surface Exclusion, Transfer, and Slow-Growth Phenotypes

The contiguous trbJ and trbK genes of RP1 were cloned individually to study their effects. Surface exclusion was conferred only by trbK and only when gene dosage was high or when trbJ was also present in cis or in trans. This suggests that in the low-copy-number RP1, surface exclusion is due to a two-gene interaction in which trbK is the dominant partner. Among surface exclusion genes, trbJ is novel in yielding a periplasmic product that is also essential for conjugal transfer. This cellular location and the disturbed membrane function that accompanies TrbJ-processing probably accounts for the retarded growth caused by trbJ + clones in Pseudomonas aeruginosa strain PAO.

The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F‐like plasmids

The effects of defined mutations in the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating-pair formation in liquid media by the transfer systems of the F-like plasmids pOX38 (F), ColB2 and R100-1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responsible for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. ColB2 transfer was more strongly affected by mutations in the heptose II-heptose III region of the LPS (rfaF) whereas R100-1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating-pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F-like plasmids (F, ColB2, R100-1) were used to complement this mutation. Unexpectedly, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.

Surface Exclusion Gene of IncI1 Plasmid R64: Nucleotide Sequence and Analysis of Deletion Mutants

We have cloned and sequenced the exc gene determining surface exclusion of IncI1 plasmid R64. R64 exc gene is highly homologous to the R144 exc gene. The R64 exc gene may encode two proteins with 220 and 147 amino acid residues by in frame reinitiation of translation, as the R144 exc gene does, while the larger protein with 220 amino acids was shown to be essential for surface exclusion function. R64 derivatives carrying deletion or insertion mutations in the exc gene transferred at a frequency similar to that of wild-type R64, indicating that the exc gene is not essential for the transfer of R64.

CD1 gene expression in human skin

In human epidermis, expression of CD1a is confined to Langerhans cells (LC), whereas CD1c expression has been observed in dendritic cells of the dermis, as well as the epidermis. In transfected fibroblasts, expression of CD1c at the cell surface appears to exclude expression of either CD1b or CD1a, despite continued transcription of the latter genes. In order to determine whether this mechanism might be operative in human skin, we have compared the expression of CD1a and CD1c on the surface of dermal and epidermal dendritic cells to their expression at the level of mRNA using a combination of dual-label immunofluorescence microscopy, northern blot hybridization, and reverse transcriptase-polymerase chain reaction (RT-PCR). By both immunofluorescence and Northern blotting, CD1c expression was observed in both dermal and epidermal cells, whereas expression of CD1a was confined largely to the epidermis. Moreover, as shown by immunomagnetic bead selection and RT-PCR, CD1a and CD1c were both expressed on epidermal LC, but were absent from other epidermal cell types. These results argue against cell surface exclusion as a mechanism for selective expression of CD1c in human dermis.

Testing protein adsorption models by off-null ellipsometry: Determination of binding constants from a single adsorption curve

The kinetics of prothrombin adsorption on planar bilayers, prepared from 40% dioleoyl phosphatidylserine and 60% dioleoyl phosphatidylcholine, are studied by off-null ellipsometry. A simple expression for the adsorption rate is derived directly from measured adsorption rates. The interpretation of this expression in terms of transport processes shows that up to a surface coverage Γ = 1.8 pmol cm −2, i.e. 50% of the maximal surface coverage Γ max, the adsorption rate appears to be regulated by diffusion-limited transport of prothrombin from the bulk solution to the adsorbing surface. The adsorption rate is proportional to the concentration difference between the bulk and the solution immediately adjacent to the surface. The latter concentration appears to be instantaneously in equilibrium with the surface excess, conforming to the classical Langmuir isotherm. For low surface coverages, below 50% of maximal adsorption, the kinetics are thus described by a simple model containing the mass transfer coefficient, the dissociation constant K d and the maximal surface coverage Γ max, With known Γ max this model allows the estimation of K d from a single adsorption curve. However, for surface coverages exceeding 60% of maximal adsorption, drastic deviations from this model are found. The intrinsic adsorption rate is substantially reduced, presumably by surface exclusion.

A microbial model for visualization of surface proteins using cryoimmobilization and high-resolution cryo-SEM

Numerous studies on high resolution visualization of surface membrane proteins have utilized colloidal gold markers in conjunction with SEM, TEM, and by freeze-fracture cytochemistry using conventional or fracture flip methods, but in each case only information on the distribution of the marker complex, and not the actual structure of individual surface protein(s) was obtained.To investigate the detection of individual surface proteins, we have used a microbial model to examine surface proteins involved in conjugation or the transfer of DNA inEnterococcus faecalis. Two surface proteins encoded by the pCF10 plasmid, aggregation substance (Asc10; globular shape) and surface exclusion protein (Sec10; helical shape), mediate the aggregation of donor and recipient cells and prevent aggregation of like donor cells, respectively. Isogenic strains of E. faecalis that constitutively express neither Asc10 or Sec10 (pWM401) or both (pINY1801) were processed by 1) conventional chemical fixation, dehydration, critical point drying with CO2, ion beam sputtering with a discontinuous platinum film (<1 nm), and examination in a Hitachi S-900 FESEM operated at low voltage (<3 keV), or 2) cryoimmobilization of cells by high pressure freezing (Balzers HPM 010), mounting in a Gatan cryoholder followed by etching for 60 minutes at −100°C, cryocoating by planar magnetron sputtering (Balzers MED 010), and examination at −100°C in the S-900 FESEM.

Structure, function, and regulation of the kilB locus of promiscuous plasmid RK2

The kil-kor regulon of the self-transmissible, broad-host-range plasmid RK2 is a unique network with eight coregulated operons. Among the genes encoded by the kil-kor regulon are trfA, which encodes the replication initiator, and several kil loci (kilA, kilB, kilC, and kilE), each of which is lethal to the host cell in the absence of appropriate negative regulatory elements encoded by the korA, korB, korC, and korE determinants. We have proposed that the functions of the kil loci are related to RK2 maintenance or host range. Here, we report the nucleotide sequence of a 2.44-kb region that includes the lethal kilB determinant. We identified the first three genes of the kilB operon (designated klbA, klbB, and klbC), and we determined by deletion analysis that the host-lethal phenotype requires klbB. The predicted amino acid sequence of the 34,995-Da klbA product reveals a potential ATP-binding fold. The klbB product is predicted to be a membrane protein with a molecular mass of 15,012 Da with homology to the RK2 KlaC membrane protein encoded by the kilA operon. The amino acid sequence of the 12,085-Da klbC product contains a perfect match to the leucine zipper motif common to eukaryotic regulatory proteins. Primer extension analysis revealed unambiguously that transcription of the kilB operon begins 46 nucleotides upstream of klbA. No transcription was initiated from the sequence previously presumed by other investigators to be the kilB promoter. The abundance of kilB transcripts is reduced in the presence of KorB, consistent with the prediction that KorB acts at the level of transcription. A degenerate KorB-binding site that contains a perfect half-palindrome overlaps the kilB promoter, but this site is insufficient for regulation by KorB. The region containing a KorB-binding site located 183 bp upstream of the transcriptional start is required for regulation by KorB, indicating that KorB acts at a distance to regulate transcription of kilB. Our studies with the mutant plasmid pRP101, a transfer-defective derivative of the RK2-like plasmid RP4, demonstrated that the kilB operon includes the conjugal transfer and surface exclusion genes of the Tra2 region. Nucleotide sequence analysis revealed that the transposon Tn7 insertion in pRP101 is located in the klbC gene, and complementation analysis showed that this mutation has a strong polar effect on the expression of genes for conjugal transfer and surface exclusion located several kilobases downstream. A klbA mutant was constructed and found to be both transfer defective and complementable, thus, demonstrating a requirement was constructed and found to be both transfer defective and complementable, thus demonstrating a requirement for klbA product in plasmid transmissibility. These results have demonstrated a role for the kilB operon in conjugal transfer. The kil-kor regulon of RK2 is the only known example of plasmid-mediated coregulation of replication and transfer.

Peculiarities of expression of the plasmid pAP42 genetic region determining ?sex? pili synthesis and the surface exclusion system in different cells ofEscherichia coli

Peculiarities of expression of the plasmid pAP42 genetic region determining ?sex? pili synthesis and the surface exclusion system in different cells ofEscherichia coli

Surface exclusion specificity of the TraT lipoprotein is determined by single alterations in a five-amino-acid region of the protein.

The TraT protein is a highly cell-surface-exposed lipoprotein specified by F-like plasmids that confers serum resistance and blocks the conjugative transfer of plasmids to cells bearing identical or closely related plasmids, a process known as surface exclusion. The TraT protein specified by the antibiotic-resistance plasmid R6-5 was purified to apparent homogeneity. When added to mating mixtures, TraT blocked the transfer of plasmids belonging to Surface Exclusion Group IV (Sfx IV) but had no significant effect on the transfer of plasmids belonging to other groups. Additionally, the purified protein has a protective effect on bacterial cells incubated in serum, indicating that it does not have to be located on the cell surface to mediate serum resistance. To localize regions of the protein that were responsible for surface exclusion specificity, the amino acid sequence of the TraT protein specified by CoIB2-K98 (Sfx II) was determined by cloning and sequencing of the corresponding gene. Comparison of the derived sequence with those of the F and R100-1 proteins indicated that surface exclusion specificity of TraT is determined by single alterations in a five-amino-acid region (residues 116-120). This was confirmed by segment swapping experiments in which the specificity of the R6-5 TraT protein (Sfx IV) was switched to that of the CoIB2-K98 protein (Sfx II). Our results suggest that the region defined by residues 116-120 is located on the external face of the outer membrane and interacts specifically with the donor cell in surface exclusion.