The physical map of the chromosome of a serogroup A strain of Neisseria meningitidis shows complex rearrangements relative to the chromosomes of the two mapped strains of the closely related species N. gonorrhoeae.

A physical map of the chromosome of N. meningitidis Z2491 (serogroup A, subgroup IV-1) has been constructed. Z2491 DNA was digested with NheI, SpeI, SgfI, PacI, BglII, or PmeI, resulting in a limited number of fragments that were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis. The estimated genome size for this strain was 2,226 kb. To construct the map, probes corresponding to single-copy genes or sequences were used on Southern blots of chromosomal DNA digested with the different mapping enzymes and subjected to CHEF electrophoresis. By determining which fragments from different digests hybridized to each specific probe, it was possible to walk back and forth between digests to form a circular macrorestriction map. The intervals between mapped restriction sites range from 10 to 143 kb in size. A total of 117 markers have been placed on the map; 75 represent identified genes, with the remaining markers defined by anonymous cloned fragments of neisserial DNA. Comparison of the arrangement of genetic loci in Z2491 with that in gonococcal strain FA1090, for which a physical map was previously constructed, revealed complex genomic rearrangements between the two strains. Although gene order is generally conserved over much of the chromosome, a region of approximately 500 kb shows translocation and/or inversion of multiple blocks of markers between the two strains. Even within the relatively conserved portions of the maps, several genetic markers are in different positions in Z2491 and FA1090.     

Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3(2).

A physical map of the chromosome of Streptomyces lividans 66 ZX7 was constructed by ordering the macrorestriction fragments generated from the genomic DNA with the restriction enzymes AseI and DraI. AseI and DraI linking cosmids (i.e., recombinant cosmids including either AseI or DraI sites) were isolated from a gene bank and used as hybridization probes against Southern transfers of pulsed-field gel electrophoresis (PFGE) restriction patterns. The DraI sites were precisely mapped by PFGE analyses of AseI-DraI double digests and hybridization with the AseI junctions. The 16 AseI and 7 DraI fragments were aligned as a single chromosome of about 8,000 kb. The data supported the interpretation that the chromosome is a linear structure. The related strain Streptomyces coelicolor A3(2) M145, recently mapped by H. Kieser, T. Kieser, and D. A. Hopwood (J. Bacteriol. 174:5496-5507, 1992), was compared with S. lividans at the level of the genomic structure by hybridizing the linking cosmids to Southern transfers of PFGE patterns. In spite of little apparent similarity in their restriction patterns, the comparison of the physical maps revealed a common structure with an identical ordering of the cosmid sequences. This conservation of the map order was further confirmed by assigning genetic markers (i.e., cloned genes and DNA elements relevant to the unstable region) to the AseI fragments.     

Physical mapping of the Mycoplasma gallisepticum S6 genome with localization of selected genes.

We report the construction of a physical map of the Mycoplasma gallisepticum S6 genome by field-inversion gel electrophoresis of DNA fragments generated by digestion of genomic DNA with rare-cutting restriction endonucleases. The size of the M. gallisepticum S6 genome was calculated to be approximately 1,054 kb. The loci of several genes have been assigned to the map by Southern hybridization utilizing specific gene probes.     

Physical and genetic map of the Methanobacterium wolfei genome and its comparison with the updated genomic map of Methanobacterium thermoautotrophicum Marburg

A physical and genetic map of the chromosome of Methanobacterium wolfei was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by digestion with NotI and NheI. The chromosome was found to be circular and 1,729 kb in size. Twenty-eight genes were mapped to specific restriction enzyme fragments by performing hybridization experiments with gene probes from various Methanobacterium strains. The genomic map obtained was compared with the updated genomic map of Methanobacterium thermoautotrophicum Marburg. In spite of major restriction pattern dissimilarities, the overall genetic organization seemed to be conserved between the genomes of the two strains. In addition, the two rRNA operons of strain Marburg were precisely mapped on the chromosome, and it was shown that they are transcribed in the same direction.     

Correlated physical and genetic map of the Bradyrhizobium japonicum 110 genome.

We describe a compilation of 79 known genes of Bradyrhizobium japonicum 110, 63 of which were placed on a correlated physical and genetic map of the chromosome. Genomic DNA was restricted with enzymes PacI, PmeI, and SwaI, which yielded two, five, and nine fragments, respectively. Linkage of some of the fragments was established by performing Southern blot hybridization experiments. For probes we used isolated, labelled fragments that were produced either by PmeI or by SwaI. Genes were mapped on individual restriction fragments by performing gene-directed mutagenesis. The principle of this method was to introduce recognition sites for all three restriction enzymes mentioned above into or very near the desired gene loci. Pulsed-field gel electrophoresis of restricted mutant DNA then resulted in an altered fragment pattern compared with wild-type DNA. This allowed us to identify overlapping fragments and to determine the exact position of any selected gene locus. The technique was limited only by the accuracy of the fragment size estimates. After linkage of all of the restriction fragments we concluded that the B. japonicum genome consists of a single, circular chromosome that is approximately 8,700 kb long. Genes directly concerned with nodulation and symbiotic nitrogen fixation are clustered in a chromosomal section that is about 380 kb long.     

Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.     

Macrorestriction Analysis of Caenorhabditis Elegans Genomic DNA

The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a ``macrorestriction mapping' procedure for Caenorhabditis elegans that we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from the C. elegans physical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is <220 kb. (2) We mapped the mes-1 gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located ~400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiency hDf6, and determined that three spontaneously derived duplications in the unc-38-dpy-5 region have very complex molecular structures, containing internal rearrangements and deletions.     

A physical and genetic map of Neisseria meningitidis B1940

A physical map of the chromosome of Neisseria meningitidis B1940 has been constructed by one- and two-dimensional pulsed-field gel electrophoresis techniques. Complete macrorestriction maps for the enzymes Nhe I (16 sites), Sgf I (13 sites), Sfi I (11 sites) and I-Ceu I (4 sites), as well as a partial restriction map for the restriction enzyme Spe I (15 of c. 28 sites) could be established. Altogether, 59 restriction sites were mapped on a single circular chromosome of 2.3 Mbp. By restriction endonuclease digestion and Southern hybridization of cloned genetic markers, 39 genetic loci were assigned to this map. Comparison with the metabolic maps of Neisseria gonorrhoeae MS11-N198 and FA1090 revealed a high degree of conservation in the arrangement of gene loci among these two species, although four out of 24 genetic loci are located at different chromosomal positions, indicating several genomic rearrangements.     

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.     

Construction of a chromosome map for the phage group II Staphylococcus aureus Ps55.

The genome size and a partial physical and genetic map have been defined for the phage group II Staphylococcus aureus Ps55. The genome size was estimated to be 2,771 kb by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI, CspI, and SgrAI. The Ps55 chromosome map was constructed by transduction of auxotrophic and cryptic transposon insertions, with known genetic and physical locations in S. aureus NCTC 8325, into the Ps55 background. PFGE and DNA hybridization analysis were used to detect the location of the transposon in Ps55. Ps55 restriction fragments were then ordered on the basis of genetic conservation between the two strains. Cloned DNA probes containing the lactose operon (lac) and genes encoding staphylococcal protein A (spa), gamma hemolysin (hlg), and coagulase (coa) were also located on the map by PFGE and hybridization analysis. This methodology enabled a direct comparison of chromosomal organization between NCTC 8325 and Ps55 strains. The chromosome size, gene order, and some of the restriction sites are conserved between the two phage group strains.     

A BlnI restriction map of the Salmonella typhimurium LT2 genome.

BlnI or AvrII (5'-CCTAGG) sites are very rare in the Salmonella typhimurium LT2 genome. BlnI was used to construct a physical map which was correlated with the genetic map by using three methods. First, Tn10 carries BlnI sites, and the extra restriction sites produced by 34 genetically mapped Tn10 insertions were physically mapped by using pulsed-field gel electrophoresis. Second, six genetically mapped Mud-P22 prophage insertions were used to assign BlnI fragments. Integration of Mud-P22 introduces 30 kb of DNA that can easily be detected by a "shift up" in all but the largest BlnI fragments. Finally, induced Mud-P22 insertions package more than 100 kb of genomic DNA adjacent to one side of the insertion. Some of the smaller BlnI fragments were localized by hybridization to a dot blot array of 52 lysates from induced Mud-P22 insertions. Of the 10 BlnI sites mapped, 6 probably occur in or near the 16S rRNA genes at about 55, 71, 83, 86, 88.5, and 89.5 min. There is one BlnI site in the 90-kb pSLT plasmid. Two additional BlnI fragments of about 7 and 4 kb have not been localized. The size of the genome was estimated as 4.78 Mb (+/- 0.1 Mb) excluding pSLT but including prophages Fels-1 and Fels-2. One BlnI fragment that maps between 55 and 59 min showed a 40-kb reduction in size in a strain cured of the approximately 40-kb Fels-2 prophage.     

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.     

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome

Abstract A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NHe I revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the Alu I-methylase six Nhe I recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior Alu I methylation, respectively. All nine Alu I-methylase/ Nhe I and 14 Nhe I restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.     

Physical map of Campylobacter jejuni TGH9011 and localization of 10 genetic markers by use of pulsed-field gel electrophoresis.

The physical map of Campylobacter jejuni TGH9011 (ATCC 43430) was constructed by mapping the three restriction enzyme sites SacII (CCGCGG), SalI (GTCGAC), and SmaI (CCCGGG) on the genome of C. jejuni by using pulsed-field gel electrophoresis and Southern hybridization. A total of 25 restriction enzyme sites were mapped onto the C. jejuni chromosome. The size of the genome was reevaluated and was shown to be 1,812.5 kb. Ten C. jejuni genetic markers that have been isolated in our laboratory were mapped to specific restriction enzyme fragments. Furthermore, we have accurately mapped one of the three rRNA operons (rrnA) and have demonstrated a separation of the 16S and 23S rRNA-encoding sequences in one of the rRNA operons.     

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 maps of the genomes of three Bacillus cereus strains.

NotI restriction maps of the chromosomes from Bacillus cereus ATCC 10876, ATCC 11778, and the B. cereus type strain ATCC 14579 have been established and compared with the previously established map of B. cereus ATCC 10987. Between 10 and 14 NotI fragments were observed, ranging from 15 to 1,300 kb, in digests of DNA from the various strains. The sizes of the genomes varied between 5.4 and 6.3 Mb. The maps were constructed by hybridization of 42 random probes, prepared from B. cereus ATCC 10987 libraries, to fragments from partial and complete NotI digests, separated by pulsed-field gel electrophoresis. Nine probes were specific for ATCC 10987 only. Probes for five B. subtilis and five B. cereus genes were also used. The NotI restriction fragment patterns of the four strains were strikingly different.     

Physical and genetic map of Streptococcus thermophilus A054.

The three restriction endonucleases SfiI, BssHII, and SmaI were found to generate fragments with suitable size distributions for mapping the genome of Streptococcus thermophilus A054. A total of 5, 8, and 24 fragments were produced with SfiI, BssHII, and SmaI, respectively. An average genome size of 1,824 kb was determined by summing the total fragment sizes obtained by digestions with these three enzymes. Partial and multiple digestions of genomic DNA in conjunction with Southern hybridization were used to map SfiI, BssHII, and SmaI fragments. All restriction fragments were arranged in a unique circular chromosome. Southern hybridization analysis with specific probes allowed 23 genetic markers to be located on the restriction map. Among them, six rrn loci were precisely located. The area of the chromosome containing the ribosomal operons was further detailed by mapping some of the ApaI and SgrAI sites. Comparison of macrorestriction patterns from three clones derived from strain A054 revealed two variable regions in the chromosome. One was associated with the tandem rrnD and rrnE loci, and the other was mapped in the region of the lactose operon.     

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae.

A physical map of the Streptococcus (Diplococcus) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.