Characterization of outer membrane proteins in Chlamydia trachomatis LGV serovar L2

We used a photoactivatable, lipophilic reagent, 3'-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies of Chlamydia trachomatis LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.     

Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.     

Construction of yeast artificial chromosome libraries by pulsed-field gel electrophoresis

Yeast artificial chromosome (YAC) libraries have been constructed from a variety of organisms using different approaches. This protocol outlines in detail the construction of YAC libraries with large inserts using size fractionation of partially digested DNA by pulsed-field gel electrophoresis.     

Isolation of a type-specific antigen from Chlamydia trachomatis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

This report describes the isolation of a type-specific antigen of a serotype A strain of Chlamydia trachomatis. The antigen could be identified in sodium dodecyl sulfate-polyacrylamide electrophoretic (SDS-PAGE) analysis of immunoprecipitates of homologously reacted lysates from Bolton-Hunter 125I-labeled elementary bodies, solubilized by sonication and treatment with Nonidet P40. The electrophoretic pattern of this precipitate revealed a peak of unique mobility that was not reproduced by heterologous or control precipitates. Immunoadsorbtion of test antigen with purified IgG fractions from homologous antisera completely removed this peak, whereas similar adsorbtion wth heterologous IgG had minimal effect. Comparison of this antigen in SDS-PAGE with protein standards revealed an approximate m.w. of 27,000.     

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues.     

Genotypic characterization of human and environmental isolates of Salmonella choleraesuis subspecies choleraesuis serovar infantis by pulsed-field gel electrophoresis.

To determine the extent of genetic diversity of Salmonella choleraesuis subspecies choleraesuis serovar Infantis and whether environmental isolates were similar or identical to human isolates, a total of 110 isolates from humans, broiler samples, egg production facilities, riverwater, sewage, and chicken meat were analyzed epidemiologically by pulsed-field gel electrophoresis. While the isolates showed 35 distinct pulsed-field profiles, none had the genotype of the human isolates. One pulsed-field profile was shared by 43 (39%) of the 110 isolates. These results indicate that relatively fewer clonal lines of S. serovar Infantis had spread widely while multiple clonal lines, including the strain involved in the outbreak, exist in Western Japan.     

Finding the sources of Korean Salmonella enterica serovar Enteritidis PT 4 isolates by pulsed-field gel electrophoresis

In previous studies, it has been reported that both S. enteritidis, the most common serotype, and S. enteritidis Phage Type 4 (SEPT 4) isolates were identified as the most prevalent PT in domestic poultry and also in humans in Korea until 2002. The aim of this study was to analyze the genetic diversity and epidemiological properties of both PT isolates, and also to trace the source of SEPT 4 isolates from domestic poultry and humans by Pulsed-field gel electrophoresis (PFGE). In order to understand the molecular epidemiologic properties of SEPT 4 isolates, which have very similar phenotypic properties to our preliminary investigations (serotyping, phage typing, large plasmids and antibiograms), PFGE analysis with XbaI enzyme was performed on the representative SEPT 4 isolates. Thirty-six SEPT 4 isolates were analyzed and differentiated with 10 pulsed-field profiles (PFP) expressing very high discriminative ability (SID: 0.921). In PFP, SEPT 4 isolates from human patients showed a perfect genetic match with those from broiler chickens and meats. Therefore, this study was able to successfully trace the major source of SEPT 4 isolates and also to determine the usefulness of the PFGE method for genetic analysis of epidemic strains.     

Differentiation of Erwinia amylovora strains by pulsed-field gel electrophoresis.

Erwinia amylovora strains, isolated from several host plants in various geographic regions during different years, were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the DNA from lysed, agar-embedded cells with rare-cutting restriction enzymes. The banding patterns obtained with enzyme XbaI digests revealed significant differences among strains from different areas. North American strains E9 and Ea-Rb, a Rubus strain, were highly divergent from other E. amylovora strains. French strains were different from central European and English strains. E. amylovora strains from central Europe and New Zealand had identical PFGE patters, as had strains from Egypt, Greece, and Turkey. PFGE of genomic DNA from American and English strains gave rise to dissimilar patterns. Patterns of some American strains resembled those from strains isolated in other parts of the world. The restriction fragment length polymorphisms observed by PFGE analysis can be used to group strains and may give hints about the course of distribution of the plant disease. From the sizes of the restriction fragments obtained, a molecular mass of approximately 4.5 Mb was calculated for the genome of E. amylovora.     

Binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) analyzed by flow cytometry

We have developed a method for quantitatively assessing binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) in several mammalian cell lines using fluorescence staining and flow cytometry. Cells were incubated with chlamydia at 4 degrees C to monitor binding; ingestion was determined by raising the temperature to 37 degrees C for 1-4 h and removing extracellular bacteria with pronase. Growth of bacteria was measured by assessing brightly stained intracellular inclusions. Fixation with methanol prior to fluorescent staining provided the most intense specific staining with minimal background, as well as preserving cell morphology. Our data reveal relatively slow ingestion of L2 by McCoy fibroblasts (maximum ingestion by 4 h) and a sizeable population of McCoy cells (30-40% of total cells) that ingest L2 but do not permit its growth under certain infectious conditions. It was possible to correlate specific histogram patterns on the flow cytometer with fluorescent microscope observations. This system provides a means of analyzing quantitative interactions between chlamydia and individual host cells.     

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

Physical and genetic mapping of the genomes of five Mycoplasma hominis strains by pulsed-field gel electrophoresis.

We present the complete maps of five Mycoplasma hominis genomes, including a detailed restriction map and the locations of a number of genetic loci. The restriction fragments were resolved by field inversion gel electrophoresis or by the contour-clamped homogeneous-electric-field system of pulsed-field gel electrophoresis. All the ApaI, SmaI, BamHI, XhoI, and SalI restriction sites (total of 21 to 33 sites in each strain) were placed on the physical map, yielding an average resolution of 26 kb. The maps were constructed using three different approaches: (i) size determination of DNA fragments partially or completely cleaved with one or two restriction enzymes, (ii) hybridization analysis with purified restriction fragments and specific probes, and (iii) use of linking clones. A genetic map was constructed by hybridization with gene-specific probes for rpoA, rpoC, rrn, tuf, gyrB, hup, ftsY, the unc operon, the genes for two M. hominis-specific antigenic membrane proteins, and one gene encoding a protein with some homology to Escherichia coli alanyl-tRNA synthetase. The positions of mapped loci were partially conserved in the five strains except in one strain in which a 300-kb fragment was inverted. The numbers and order of mapped restriction sites were only partly conserved, and this conservation was restricted to certain regions. The gene order was compared with the gene order established for other bacteria and was found to be identical to that of the phylogenetically related Clostridium perfringens. The genome size of the M. hominis strains varied from 704 to 825 kb.     

Identification by sequence analysis of two-site posttranslational processing of the cysteine-rich outer membrane protein 2 of Chlamydia trachomatis serovar L2.

The 60,000-molecular-weight cysteine-rich outer membrane protein (OMP2) from Chlamydia trachomatis participates in the disulfide-mediated outer-membrane organization unique to this organism. In addition, this protein is a primary focus of the host immune response. We cloned and sequenced the gene for OMP2 from C. trachomatis serovar L2. A lambda gt11 recombinant that expressed an antigenic portion of this protein was selected by antibody screening and provided a probe for the selection in lambda 1059 of a clone containing the entire gene. DNA sequencing of this clone identified one open reading frame of 1,641 base pairs, starting with a methionine codon and coding for a polypeptide with a molecular weight of 58,792. Amino-terminal protein sequencing and analysis of the translated DNA sequence demonstrated that processing at alternate signal peptide cleavage sites accounts for the molecular-weight polymorphism of this protein. The mature proteins had a net positive charge and contained 24 cysteine residues.     

Genome analysis ofPropionibacterium freudenreichii by pulsed-field gel electrophoresis

Preparations of intact genomic DNA from 23 strains ofPropionibacterium freudenreichii were compared by digestion with restriction endonucleases and subsequent transverse alternating field electrophoresis (TAFE). Seven restriction enzymes,AsnI,DraI,HpaI,SnaBI,SpeI,SspI, andXbaI, produced DNA fragments useful for strain comparisons. A characteristic restriction fragment pattern was identified for 18 of the 23 strains. Estimates for the genome size of theP. freudenreichii strains ranged from 1.6×10⁶ to 2.3×10⁶ base pairs based on the sum of fragment sizes obtained with restriction digests. Restriction endonuclease patterns resolved by TAFE are useful for strain identification.     

Construction of physical maps of the Hor1 locus of two barley cultivars by pulsed field gel electrophoresis.

Summary In order to construct a physical map of the Hor1 locus of barley (Hordeum vulgare) high molecular weight DNA was prepared from leaf mesophyll protoplasts. Seventeen different restriction endonucleases containing CpG or CpXpG motifs in their recognition sequences were tested and ten proved useful for the generation of high molecular weight DNA fragments. Physical maps of the Hor1 region of the barley cultivars IGRI and FRANKA spanning a distance of 370 and 430 kb respectively were constructed. The maps include sites of nine restriction endonucleases in IGRI and of eight in FRANKA. The maximal extent of the Hor1 locus could be limited to a 135 kb DNA fragment occurring in both cultivars. The differences in arrangement of restriction sites and in fragment lengths reveal major differences in the Hor1 flanking region in the two cultivars. The location of a CpG island, however, is highly conserved in both cultivars and reflects similarities to the organization of mammalian genomes.     

Genome size of Myxococcus xanthus determined by pulsed-field gel electrophoresis.

Genomic DNA of the myxobacterium Myxococcus xanthus was digested with the rare cutting restriction endonuclease AseI or SpeI, and the restriction products were separated by pulsed-field gel electrophoresis. Transposons Tn5-132 and Tn5 lac, which contain AseI restriction sites, were used to determine the number of restriction fragments in each band. The size of the genome was determined by adding the molecular sizes of the restriction products. The genomes of strains DK101, MD2, and DZF1 have identical restriction patterns and were estimated to be 9,454 +/- 101 kilobase pairs from the AseI digestions and 9,453 +/- 106 kilobase pairs from the SpeI digestions. DK1622, which was derived from DK101 by treatment with UV light, has suffered a 220- to 222-kilobase-pair deletion that removed an AseI and an SpeI restriction site. The deleted DNA may consist exclusively of Mx alpha-associated sequences.     

Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis.

Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect.     

Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis.

A method for constructing yeast artificial chromosome (YAC) libraries with large insert sizes is reported. High molecular weight human DNA was partially digested with EcoRI and cloned in the vector pYAC4. When unfractionated DNA was used, the mean YAC size was 120kb. Fractionation by pulsed-field gel electrophoresis using a 'waltzer' apparatus to remove small DNA fragments increased the mean YAC size to congruent to 220kb or congruent to 370kb depending on the fractionation conditions. Ligated DNA prepared by this method was stable at 4 degrees C and routinely yielded transformation efficiencies of greater than 700 colonies/micrograms. It should be possible to extend the method to produce even larger inserts and to use high molecular weight DNA from any source.     

Determination of Wolbachia genome size by pulsed-field gel electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.     

Analysis of large DMA from soybean (Glycine max L. Merr.) by pulsed-field gel electrophoresis

The technique of pulsed-field gel electrophoresis (PFE) has been used to study chromosomal regions and entire genomes of several organisms. Techniques are presented for the isolation of high molecular weight DNA from embedded soybean protoplasts and the conditions for separating large DNA fragments using PFE. Digestion was detected by Southern hybridization using single copy nodulin clones. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.