Rapid expansion of the physical and genetic map of the chromosome of Clostridium perfringens CPN50

Abstract

The physical map of the 3.6-megabase chromosome of Clostridium perfringens CPN50 was extended by positioning sites for the endonucleases SfiI and I-CeuI, and in parallel, the gene map was expanded by using a genome scanning strategy. This involved the cloning and sequencing of random chromosomal fragments, identification of the functions of the putative genes by database searches, and then hybridization analysis. The current gene map comprises almost 100 markers, many of which encode housekeeping functions while others are involved in sporulation or pathogenesis. Strikingly, most of the virulence genes were found to be confined to a 1,200-kb segment of the chromosome near oriC, while the pleiotropic regulatory locus, virRS, was situated toward the putative replication terminus. A comparison of the gene maps of three endospore-forming bacilli, C. perfringens, Clostridium beijerinckii, and Bacillus subtilis, revealed a similar order and distribution of key sporulation and heat shock genes which might reflect an ancient evolutionary relationship.Clostridium perfringens is a spore-forming, gram-positive anaerobe commonly found in the lower intestinal tracts of humans and other mammals as well as in soil and sewage. C. perfringens has been shown to cause a variety of diseases ranging in severity from the frequently fatal gas gangrene to a mild but common form of food poisoning (18,32). Clinical isolates can be classified into five serotypes, A to E, on the basis of their production of the four lethal typing toxins, the ␣-, ␤-, ε-, and -toxins (18,26,32). In addition to the typing toxins, most strains of C. perfringens produce a large variety of other toxins and hydrolytic enzymes, such as perfringolysin O, or -toxin, and collagenase, or -toxin, (23,25,33), that are likely to play a significant role in pathogenesis.A significant step forward in understanding the molecular genetics of this medically important anaerobe was made some years ago when a physical map of the chromosome of the paradigm strain of C. perfringens, CPN50, was constructed with six restriction enzymes and the positions of 28 genes were established (6). Subsequently, the extent of genomic diversity among the various serovars was assessed, three new markers were added, and the variation in the toxin gene repertoire was examined (9).Although a number of genes have been cloned recently from the clostridia, the rate of identification of new genetic markers has been relatively low. Consequently, in an attempt to rapidly expand the information content of the genome map, we have applied the technique of genome scanning in which random chromosomal fragments are cloned and sequenced and their putative functions are identified by means of database searches. These are then positioned on the physical map by means of hybridization. This strategy was particularly productive and not only led to the identification of 54 new loci but also generated a number of interesting leads for research in pathogenesis because several potential virulence genes were isolated. MATE...

Physical and genetic map of the Clostridium acetobutylicum ATCC 824 chromosome

“…A similar orientation of the rrn operons has already been described for many bacteria, e.g., Escherichia coli (2), B. subtilis (39), C. perfringens (9,29), and Lactococcus lactis (31,49). The presence of doublets or triplets of rrn operons is less frequent but has been described in C. perfringens (29), C. beijerinckii (55), B. subtilis (39), and Streptococcus thermophilus (41). The B. subtilis chromosome carries both a triplet (rrnGHI) and a doublet (rrnJW) of rrn operons (39).…”

“…The genetic map of C. acetobutylicum presented in this study is the fourth for endospore-forming bacteria and follows those for B. subtilis (4), C. beijerinckii (55), and C. perfringens (9,29). We have extended the comparison (29) between these maps and the map of C. acetobutylicum ATCC 824 (Fig.…”

Evaluation of Clostridium ljungdahlii DSM 13528 reference genes in gene expression studies by qRT-PCR

“…Acetogens fix inorganic carbon (CO and CO 2 ) for survival through the Wood-Ljungdahl pathway, in which formate tertrahydrofolate ligase is an essential enzyme. Although fotl is not widely used as a reference gene for normalization, it is considered to be a putative housekeeping gene in acetogens (26,27). Additionally, in this study, the expression of fotl under all five conditions was relatively stable in C. ljungdahlii DSM 13528.…”

Physical map of the genome of Planctomyces limnophilus, a representative of the phylogenetically distinct planctomycete lineage

“…Although the reported physical map of Chlamydia trachomatis does not show a putative origin of replication (2), if the origin is tentatively placed at the single rrn locus (the two are often linked in organisms with only one rrn operon), then the dnaK gene appears to also be opposite to the origin. The tuf genes of E. coli (1), B. subtilis (41), R. sphaeroides (33), M. pneumoniae (38), M. genitalium (27), and C. perfringens (14) are similarly centrally located, whereas they are distal in P. limnophilus, as is also found for Mycoplasma hominis strains (17).…”

“…However, some comparisons between the genome of P. limnophilus and those of selected other bacteria can be drawn. For example, in P. limnophilus, rpoD is located close to the putative origin, as in E. coli (1), whereas rpoD in B. subtilis (41), Rhodobacter sphaeroides (33), and Clostridium perfringens (14) is found almost opposite the origin. rpoD in P. limnophilus is closely linked to rrl, as is the case with Campylobacter species (34).…”