Conformational change in the herpes simplex single-strand binding protein induced by DNA.

Protease digestion of the herpes simplex virus type 1 major single-strand DNA binding protein ICP8 showed that the cleavage patterns observed in the presence and absence of single-stranded DNA oligonucleotides are substantially different with protection of cleavage sites between amino acids 293 and 806 observed in the presence of oligonucleotide. Experiments using ICP8 modified with fluorescein-5-maleimide (FM) showed that the fluorescence signal exhibited increased susceptibility to antibody quenching and a significant decrease in polarization of the FM fluorescence was observed in the presence compared to the absence of oligonucleotide. Taken together, these results indicate that ICP8 undergoes a conformational change upon binding to single-stranded DNA.

Bacteriophage T4 gene 41 helicase and gene 59 helicase-loading protein: a versatile couple with roles in replication and recombination.

Bacteriophage T4 uses two modes of replication initiation: origin-dependent replication early in infection and recombination-dependent replication at later times. The same relatively simple complex of T4 replication proteins is responsible for both modes of DNA synthesis. Thus the mechanism for loading the T4 41 helicase must be versatile enough to allow it to be loaded on R loops created by transcription at several origins, on D loops created by recombination, and on stalled replication forks. T4 59 helicase-loading protein is a small, basic, almost completely alpha-helical protein whose N-terminal domain has structural similarity to high mobility group family proteins. In this paper we review recent evidence that 59 protein recognizes specific structures rather than specific sequences. It binds and loads the helicase on replication forks and on three- and four-stranded (Holliday junction) recombination structures, without sequence specificity. We summarize our experiments showing that purified T4 enzymes catalyze complete unidirectional replication of a plasmid containing the T4 ori(uvsY) origin, with a preformed R loop at the position of the R loop identified at this origin in vivo. This replication depends on the 41 helicase and is strongly stimulated by 59 protein. Moreover, the helicase-loading protein helps to coordinate leading and lagging strand synthesis by blocking replication on the ori(uvsY) R loop plasmid until the helicase is loaded. The T4 enzymes also can replicate plasmids with R loops that do not have a T4 origin sequence, but only if the R loops are within an easily unwound DNA sequence.

UvsW protein regulates bacteriophage T4 origin-dependent replication by unwinding R-loops.

The UvsW protein of bacteriophage T4 is involved in many aspects of phage DNA metabolism, including repair, recombination, and recombination-dependent replication. UvsW has also been implicated in the repression of origin-dependent replication at late times of infection, when UvsW is normally synthesized. Two well-characterized T4 origins, ori(uvsY) and ori(34), are believed to initiate replication through an R-loop mechanism. Here we provide both in vivo and in vitro evidence that UvsW is an RNA-DNA helicase that catalyzes the dissociation of RNA from origin R-loops. Two-dimensional gel analyses show that the replicative intermediates formed at ori(uvsY) persist longer in a uvsW mutant infection than in a wild-type infection. In addition, the inappropriate early expression of UvsW protein results in the loss of these replicative intermediates. Using a synthetic origin R-loop, we also demonstrate that purified UvsW functions as a helicase that efficiently dissociates RNA from R-loops. These and previous results from a number of studies provide strong evidence that UvsW is a molecular switch that allows T4 replication to progress from a mode that initiates from R-loops at origins to a mode that initiates from D-loops formed by recombination proteins.

Bacteriophage T4 proteins replicate plasmids with a preformed R loop at the T4 ori(uvsY) replication origin in vitro.

Bacteriophage T4 DNA replication proteins catalyze complete unidirectional replication of plasmids containing the T4 ori(uvsY) replication origin in vitro, beginning with a preformed R loop at the position of the origin R loop previously identified in vivo. T4 DNA polymerase, clamp, clamp loader, and 32 protein are needed for initial elongation of the RNA, which serves as the leading-strand primer. Normal replication is dependent on T4 41 helicase and 61 primase and is strongly stimulated by the 59 helicase loading protein. 59 protein slows replication without the helicase. As expected, leading-strand synthesis stalls prematurely in the absence of T4 DNA topoisomerase. A DNA unwinding element (DUE) is essential for replication, but the ori(uvsY) DUE can be replaced by other DUE sequences.

The R-type pyocin of Pseudomonas aeruginosa C is a bacteriophage tail-like particle that contains single-stranded DNA.

Pseudomonas aeruginosa R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from P. aeruginosa C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digested P. aeruginosa C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18 P. aeruginosa strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of P. aeruginosa C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonal prt genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from P. aeruginosa C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of P. aeruginosa C.

Identification of a region of the herpes simplex virus single-stranded DNA-binding protein involved in cooperative binding.

We have identified a region of the herpes simplex virus major DNA-binding protein (ICP8) which is involved in cooperative binding to single-stranded DNA. This has been accomplished by analysis of ICP8 which was covalently modified by reaction with the extrinsic fluorophore fluorescein-5-maleimide (FM). Reaction conditions which result in the incorporation of 1 mol of FM per mol of ICP8 have been established. The binding properties of the modified protein were analyzed by polyacrylamide gel shift analysis with model oligonucleotides. This analysis indicates that while intrinsic binding is similar to that observed with unmodified protein, the cooperative binding of the modified protein to single-stranded DNA is significantly altered. Helix-destabilizing assays, whose results are a reflection of cooperative binding, also indicate that this property of ICP8 is decreased upon modification with FM. Mapping of the site of modification by cyanogen bromide cleavage and peptide sequencing has shown that the major site of modification is cysteine 254. This position in the primary structure of ICP8 is distinct from the regions previously shown to be involved in the interaction of this protein with single-stranded DNA.

Selection and immunochemical analysis of lipooligosaccharide mutants of Neisseria gonorrhoeae.

The identification of enterobacterial mutants that contain alterations in the lipopolysaccharide (LPS) oligosaccharide core structure facilitated the development of the model of the physicochemical and immunochemical structures of enteric LPS. Results of recent immunochemical studies have suggested that the structural model of the lipooligosaccharides (LOSs) of Neisseria gonorrhoeae may differ from the enteric LPS model. The difficulties in the analysis of the wild-type gonococcal LOS have precluded understanding of the precise nature of the LOS structure. This study was undertaken to isolate a series of mutants of N. gonorrhoeae 1291 that had sequential saccharide deletions in the LOS. Results of preliminary studies suggested that the pyocin, designated pyocin C, allowed selection of gonococci with such mutant LOS structures. Results also indicated that the receptor for pyocin C binding was an LOS component. Pyocin C selection led to the isolation of five strains with LOS patterns on sodium dodecyl sulfate-polyacrylamide gels which differed from the LOS of parent strain 1291. In this system, the Mr of the parent LOS was 4,715, while the LOSs from the mutant strains demonstrated progressive saccharide deletions, with Mrs of 4,230, 4,089, 3,627, 3,262, and 3,197. Protein patterns of these mutants on sodium dodecyl sulfate-polyacrylamide gels were qualitatively similar to those of the parent strains. Results of studies with five monoclonal antibodies specific for neisserial LOS indicated that shared as well as unique epitopes were present on the mutant LOSs. Results of ketodeoxyoctonate analysis of the mutant LOSs indicated that the majority of the ketodeoxyoctonate residues may be substituted on C-4 or C-5. Chemical and immunological analysis of such LOS mutants should expedite the development of the model for the structure of gonococcal LOS.

Capnocytophaga species: increased resistance of clinical isolates to serum bactericidal action.

Capnocytophaga, a newly recognized genus of capnophilic gram-negative bacilli, is part of the normal oral flora. The capacity of Capnocytophaga to cause sepsis and local infections in both immunocompromised and nonimmunocompromised hosts has been documented. Given the recognition that serum resistance may contribute to the virulence of some gram-negative bacteria, we attempted to define the serum sensitivity of clinical isolates of Capnocytophaga from blood and other sites of infection. Whereas nine of nine isolates from human subgingival plaque showed greater than 95% loss of viability under standardized assay conditions, nonoral isolates exhibited variable serum sensitivity. Six of six isolates from blood showed considerable serum resistance (mean survival, 59.7% +/- 38.3%; range, 14.4%-113.3%). Comparison of the electrophoretic mobilities of lipopolysaccharides (LPSs) from sensitive and resistant strains revealed reduced LPS heterogeneity and lower apparent molecular weight among serum-resistant strains. Thus, serum resistance, possibly influenced by LPS structure, may be an important factor contributing to the pathogenic potential of Capnocytophaga spp.

Antigenic diversity of lipooligosaccharides of nontypable Haemophilus influenzae.

The lipooligosaccharides (LOS) of nontypable Haemophilus influenzae are an antigenically heterogeneous group of macromolecules. Immunodiffusion and enzyme-linked immunosorbent assay inhibition studies with phenol-water-extracted LOS and absorbed antisera specific for the oligosaccharide portion of the LOS identified six LOS strain-specific antigens. To facilitate screening large numbers of strains to search for LOS antigenic heterogeneity, a system utilizing proteinase K whole cell digests in Western blots was developed. Seventy-two nontypable H. influenzae LOS extracts were analyzed in this Western blot assay. Thirty-seven of these extracts could be segregated into 10 antigenically distinct LOS groups based on immunologic recognition by one or more of the rabbit antisera. Thirty-five of the strains did not contain these LOS antigens. These results demonstrate that antigenic differences exist among the LOS of nontypable H. influenzae strains, and this heterogeneity has the potential to be used to establish an LOS-based serogrouping system.

Identification of a 16,600-dalton outer membrane protein on nontypeable Haemophilus influenzae as a target for human serum bactericidal antibody.

A 16,600-D outer membrane protein is present in all strains of Haemophilus influenzae and antibodies to this protein are present in human serum. This study was designed to assess the role of this outer membrane protein (P6) in nontypeable H. influenzae as a target for human serum bactericidal antibody. P6 was isolated and coupled to an affinity column. Depleting normal human serum of antibodies to P6 by affinity chromatography resulted in reduced bactericidal activity of that serum for nontypeable H. influenzae. Immunopurified antibodies to P6 from human serum were bactericidal. Finally, preincubation of bacteria with a monoclonal antibody that recognizes a surface epitope on P6, inhibited human serum bactericidal killing. Taken together, these experiments establish that P6 is a target for human bactericidal antibodies. This observation provides evidence that P6 plays a potentially important role in human immunity to infection by nontypeable H. influenzae.

Identification of a specific epitope of Haemophilus influenzae on a 16,600-dalton outer membrane protein.

A mouse monoclonal antibody that recognizes an epitope on a 16,600-dalton outer membrane protein was developed to nontypable Haemophilus influenzae. This epitope was present on all 115 isolates of H. influenzae tested, including typable and nontypable strains. Screening of 89 strains of other bacteria demonstrated that this epitope is a highly specific marker for H. influenzae because the epitope was absent in virtually all other bacterial species tested. Western blot assays were performed with two normal human serum samples and convalescent-phase serum from an adult with bacteremia due to nontypable H. influenzae. Antibody to the 16,600-dalton outer membrane protein was present in all three human serum samples.

Antigenic heterogeneity of lipid A of Haemophilus influenzae.

The chemical structure and biologic function of the lipid A portion of lipopolysaccharide are not identical among gram-negative bacteria. This study indicates that antigenically heterogeneous lipid A exists among strains of Haemophilus influenzae. An immunoglobulin G3 murine monoclonal antibody, 3D2, produced against a nontypable H. influenzae strain 3524 has specificity for a site on the lipid A portion of the H. influenzae lipopolysaccharide. With the Western blot and immunodot assay, 3D2 recognized this lipid A determinant on 14 of 24 (58%) of strains of nontypable H. influenzae and in 51 of 95 (54%) strains of H. influenzae type b. This lipid A epitope has a high degree of specificity for H. influenzae, since it is not present on the lipid A of 39 gram-negative strains from 14 non-Haemophilus species. In addition, studies of 36 strains of six Haemophilus species other than H. influenzae and 8 strains of 4 species of Actinobacillus did not contain the 3D2 epitope. Enzyme-linked immunosorbent assay analysis with a kinetic assay and enzyme-linked immunosorbent assay inhibition confirmed the antigenic heterogeneity of H. influenzae lipid A. Thin-layer chromatography demonstrated that the 3D2 epitope is associated with a chloroform-soluble lipid moiety in the lipid A. Fluorescent antibody analysis of H. influenzae indicated that the epitope is on the cell surface. The monoclonal antibody was not bactericidal for strain 3524, and it did not inhibit the bactericidal action of normal human serum against the same strain. These studies demonstrate that the lipid As of H. influenzae are antigenically heterogeneous.

Fimbriation of Haemophilus species isolated from the respiratory tract of adults.

Twenty-two clinical isolates of Haemophilus species were studied within two passages of their original isolation for the presence of fimbriae by negative-staining electron microscopy. Six isolates were identified as fimbriated, including three strains of nontypable Haemophilus influenzae, one strain of Haemophilus parainfluenzae, and two strains of Haemophilus haemolyticus. In fresh isolates of fimbriated strains of nontypable H. influenzae, approximately 40%-50% of cells had fimbriae; after five passages in vitro, less than 1% of the cells had fimbriae. Thus, a variety of Haemophilus species that colonize the human respiratory tract can be fimbriated, and this fimbriation is rapidly lost on passage in vitro.

A subtyping system for nontypable Haemophilus influenzae based on outer-membrane proteins.

Outer-membrane proteins from 48 isolates of nontypable Haemophilus influenzae from adults and children were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the biotypes were determined. Each strain had two principal outer-membrane proteins in the 32,000-42,000 molecular-weight range. Eight subtypes were identified based on the differences in molecular weights of the two principal outer-membrane proteins. The lower molecular-weight protein of those two major proteins was heat modifiable. There was no definite relationship between subtype and biotype. Future studies on the pathogenesis and epidemiology of infections due to nontypable H. influenzae must take into account the differences in outer-membrane proteins among various strains. A subtyping system based on major outer-membrane proteins may provide a basis for such studies.