Physical and genetic map of the Listeria monocytogenes EGD serotype 1/2a chromosome

Listeria monocytogenes is a facultative intracellular pathogen responsible for both invasive and non-invasive food-borne illness in animals and humans. In this study, macrorestriction analysis following pulsed-field gel electrophoresis was used to show that Listeria monocytogenes serovar 1/2a strain EGD has a single chromosome containing eight NotI fragments of 1100, 850, 365, 320, 275, 40, 30 and 20 kb in size and 11 AscI fragments of 860, 470, 410, 360, 320, 250, 110, 80, 50, 30 and 20 kb. The total genome therefore comprises 3000 +/- 50 kb. The creation of a physical and genetic map of the Listeria genome was achieved by generating NotI linking clones and their use in subsequent hybridisation analysis. Using isogenic mutants harbouring additional artificial NotI restriction sites, we were able to precisely map the positions of all currently known virulence genes on the chromosome.     

Physical map of the Myxococcus xanthus chromosome.

The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social and developmental behaviors were examined by using pulsed field gel electrophoresis. Intact genomic DNA was digested with AseI into 16 restriction fragments, which were separated by contour-clamped homogeneous electric field electrophoresis, purified, and radiolabeled. Each AseI fragment was hybridized to SpeI-digested DNA and to an M. xanthus genomic library contained in yeast artificial chromosomes. Some SpeI restriction fragments and yeast artificial chromosome clones contained AseI sites and hybridized with two different AseI restriction fragments, providing evidence for the juxtaposition of these AseI restriction fragments in the chromosome. The deduced AseI physical map is circular, suggesting that this bacterium contains a single, circular chromosome. Transposable elements shown by transduction to be in or near genes of interest were located on specific AseI restriction fragments by restriction analysis and Southern hybridization. Most AseI restriction fragments contained genes involved in social and developmental behaviors.     

Physical map of the Bacillus cereus chromosome.

A physical map of the Bacillus cereus chromosome has been constructed by aligning 11 NotI fragments, ranging in size from 200 to 1,300 kilobases. The size of the chromosome is about 5.7 megabases. This is the first Bacillus genome of which a complete physical map has been described.     

Physical map of the Treponema denticola circular chromosome.

A physical map of the Treponema denticola ATCC 33520 genome was constructed by pulsed-field gel electrophoresis and DNA hybridization. The organism possesses a single, circular chromosome of approximately 3.0 Mbp and a 2.6-kbp circular plasmid, pTD1. The physical map of the A+T-rich genome was constructed with the rare-cutting restriction enzymes AscI, NotI, and SrfI, which have 8-bp G+C-rich recognition sites. The genes flgE, tdpA, and prtB encoding the flagellar hook protein, a 53-kDa immunogenic protein, and chymotrypsinlike protease, respectively, were located on the map. This treponeme was found to have two copies of each of the rRNA genes, as has been found to be the case for both Treponema phagedenis and Treponema pallidum.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Physical map of the linear chromosome of Streptomyces griseus.

The chromosomal DNA of Streptomyces griseus 2247 (a derivative of strain IFO3237) was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestion with AseI and DraI gave 15 and 9 fragments, respectively, the total sizes of which were 7.8 Mb. All the AseI and DraI fragments were aligned on a linear chromosome map by using linking plasmids and cosmids. PFGE analysis of the intact chromosome also showed a linear DNA band of about 8 Mb. Detailed physical maps of both terminal regions were constructed; they revealed the presence of a 24-kb terminal inverted repeat on each end. PFGE analysis with and without proteinase K treatment suggested that each end of the chromosome carries a protein molecule.     

Physical map of the coliphage 186 chromosome

Summary A physical map of coliphage 186 was established by cloning various restriction fragments of 186 DNA into the plasmid vector pBR322 and determining the genes encoded on each fragment by marker rescue experiments using a range of 186 mutant phage.     

Physical map of the Brucella melitensis 16 M chromosome.

We present the first restriction map of the Brucella melitensis 16 M chromosome obtained by Southern blot hybridization of SpeI, XhoI, and XbaI fragments separated by pulsed-field gel electrophoresis. All restriction fragments (a total of 113) were mapped into an open circle. The main difficulty in mapping involved the exceedingly high number of restriction fragments, as was expected considering the 59% G + C content of the Brucella genome. Several cloned genes were placed on this map, especially rRNA operons which are repeated three times. The size of the B. melitensis chromosome, estimated as 2,600 kb long in a previous study, appeared longer (3,130 kb) by restriction mapping. This restriction map is an initial approach to achieve a genetic map of the Brucella chromosome.     

Physical and antigenic heterogeneity in the flagellins of Listeria monocytogenes and L. ivanovii

Listeria monocytogenes serotypes 4a, 4b and 7, and L. ivanovii, all grown at 20 degrees C, were negatively stained and examined by electron microscopy. Crude extracts of the cell surface of L. monocytogenes serotypes 1/2b, 3b, 3c, 4a, 4b, 4d and 7 and of L. ivanovii (all grown at 20 degrees C) were examined by SDS-PAGE and Western blotting using (i) affinity-purified polyclonal monospecific antibody, and (ii) monoclonal antibody, each raised against 29 kDa flagellin of serotype 4b. No flagella were seen on serotype 7 by electron microscopy and no flagellin was detected in crude cell surface extracts of serotype 7 either in silver-stained gels or in Western blots. The monospecific polyclonal antibody detected flagellins of approximate molecular mass 29 kDa in each of the seven flagellate strains including L. ivanovii. The monoclonal antibody detected 29 kDa flagellin in serotypes 1/2b, 3b, 4a, 4b and 4d, but not the flagellins of serotype 3c or L. ivanovii, which had a slightly lower molecular mass. Following prolonged electrophoresis of crude flagellar extracts the 29 kDa complex was resolved into three closely migrating bands. In a heterologous system using serotype 1/2b crude flagellar extract, all three bands were detected using the polyclonal antibody whereas only two bands were detected by the monoclonal antibody. It is concluded that polyclonal anti-flagellin antibodies are not useful tools with which to distinguish serotypes of L. monocytogenes sensu lato in immunoblotting, but that differences can be determined using a monoclonal antibody directed against particular components of the flagellar complex. These differences did not fully correspond to those anticipated from results of agglutination tests.     

Chemiluminescence by Listeria monocytogenes.

Listeria monocytogenes cells suspended in brain heart infusion broth or in carbonated saline solution emitted light (chemiluminescence) that could be detected by a liquid scintillation spectrometer. This chemiluminescence was inhibited by superoxide dismutase and catalase but not by the hydroxyl radical scavengers mannitol and benzoate; it was also dependent upon and proportional to the carbonate ion concentration in the medium. Organisms suspended in carbonated saline solution which had ceased to chemiluminesce immediately began to chemiluminesce again when acetaldehyde was added but not when glucose, sucrose, or xanthine was added. Acetaldehyde-induced chemiluminescence was inhibited by suproxide dismutase and catalase but not by allopurinol. Our data indicate that the superoxide anion, hydrogen peroxide, and the carbonate ion are involved in chemiluminescence by L. monocytogenes. Chemiluminescence is apparently initiated by the extracellular generation of superoxide anon by this organism. The mechanism for the production of the superoxide anion is not known, but xanthine oxidase does not appear to be involved.     

Physical and genetic map of the Methanococcus voltae chromosome

A physical map of the Methanococcus voltae chromosome was constructed on the basis of restriction mapping and cross-hybridization experiments, employing total and partial digests obtained with rarely cutting restriction enzymes. On the basis of the sum of the fragment sizes of digests with seven enzymes the chromosome length was calculated to be approximately 1900 kb. The derived map is circular. Hybridization of gene probes to mapped restriction fragments has led to a genetic map of genes for structural RNAs as well as proteins, including enzymes involved in the methanogenic pathway.     

Physical map of the chromosome of the apple proliferation phytoplasma

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with BssHII, SmaI, MluI, and ApaI restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the fus and tuf genes, and three other genes were placed on the map. Genome sizes and BssHII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.     

Plasmids in Listeria monocytogenes and other Listeria species.

One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance.     

Physical and genetic map of the Serpulina hyodysenteriae B78T chromosome.

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.     

Lipoteichoic acid from Listeria monocytogenes.

A lipoteichoic acid (LTA) was extracted from Listeria monocytogenes (serotype 1) by phenol-water partition and isolated by gel-filtration chromatography. The LTA exhibited amphiphilic properties by changes in gel-filtration mobility in the presence of detergent buffers and after mild base hydrolysis. In a hemagglutination assay, Listeria LTA bound antibody prepared against a known LTA from Streptococcus spp. Listeria LTA inhibited the binding of anti-LTA antibody to a Lactobacillus LTA in a hemagglutination inhibition assay. The Listeria LTA contained glucose, galactose, fatty acids, glycerol, and phosphate with molar ratios of 0.05, 0.07, 0.21, 0.94, and 1.0 to phosphate, respectively. Adjacent glycerols were linked between the C-1 and C-3 positions by phosphodiesters (structural type 1). The average chain length was 19 +/- 2 (standard deviation) glycerol-phosphate repeating units. Approximately one glycosyl side chain was present per LTA molecule. The side chain was a galactose-containing disaccharide. The lipid portion of the LTA was a galactose- and glucose-containing glycolipid which may have been a phosphoglycolipid, but the structure was not confirmed. Major fatty acids of LTA and the glycolipid were 17:anteiso, 15:anteiso, 16:iso, 16:n, and 18:n. L. monocytogenes contained cell wall products typical of gram-positive bacteria which is in contrast to the reports by others of the presence of lipopolysaccharides from L. monocytogenes.