Genetic diversity among Botulinum Neurotoxin-producing clostridial strains.

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.

Ultrasensitive, direct detection of a specific DNA sequence of Bacillus anthracis in solution.

A very fast and ultrasensitive method has been developed for the detection and quantitation of specific nucleic and sequences of bacterial origin in solution. The method is based on a two-color, single fluorescent molecule detection technique developed in our laboratory. The technique was applied to the detection of Bacillus anthracis DNA in solution.

Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis.

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC(1), vrrC(2), vrrB(1), vrrB(2), and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes.

The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, "shotgun" cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a "pathogenicity island," defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.

Molecular diversity in Bacillus anthracis.

Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. We have identified nine novel variable number tandemly repeated loci from previously known amplified fragment length polymorphism markers or from the DNA sequence. In combination with the previously known vrrA locus, these markers provide discrimination power to genetically characterize B. anthracis isolates. The variable number tandem repeat (VNTR) loci are found in both gene coding (genic) and non-coding (non-genic) regions. The genic differences are 'in frame' and result in additions or deletion of amino acids to the predicted proteins. Due the rarity of molecular differences, the VNTR changes represent a significant portion of the genetic variation found within B. anthracis. This variation could represent an important adaptive mechanism. Marker similarity and differences among diverse isolates have identified seven major diversity groups that may represent the only world-wide B. anthracis clones. The lineages reconstructed using these data may reflect the dispersal and evolution of this pathogen.

Sequence, assembly and analysis of pX01 and pX02.

Bacillus anthracis plasmids pX01 and pX02, harboured by the Sterne and Pasteur strains, respectively, have been sequenced by random 'shotgun' cloning and high throughout sequence analysis. These sequences have been assembled (Sequencher) to generate a circulate pX01 plasmid containing 181 656 bp and a single linear (gapped) pX02 contig containing at least 93.479 bp. Initial annotation suggests that the two plasmids combined contain at least 200 potential open reading frames (ORFs) with < 40% having significant similarity to sequences registered in open databases. Collectively, only 118 566 bp of the pX01 DNA (65%) represent predicted coding regions. This value is similar to published gene densities for other plasmids and is indicative of the larger intergenic spaces in plasmids vs those found in the chromosomes of the parental microbes (85-93% gene density). A 70 kbp region including the toxin genes (cya, lef and pag) is distinct from the remainder of the pX01 sequence: (1) it has a lower gene density (58 vs 70%) than the remaining 111 kbp; (2) it contains all but one of the co-regulated transcriptional fusions identified by transposon mutagenesis (Hoffmaster & Koehler 1997) and (3) it contains a significantly higher proportion of positive BLAST scores (62 vs 20%) for putative ORFs. These data suggest different origins for the two regions of pX01.

Heritable genetic alterations in a xeroderma pigmentosum group G/Cockayne syndrome pedigree.

A search for genetic alterations within the XPG gene has been conducted on skin and blood cells cultured from a newly characterized xeroderma pigmentosum (XP) patient (XP20BE). This patient is the ninth known case that falls into the extremely rare XP complementation group G. Four genetic markers within the XPG gene (including two polymorphisms) demonstrated the Mendelian distribution of this gene from the parents to the patient and to an unaffected sibling. The patient (XP20BE) inherited a G to T transversion from his father in exon 1 of the XPG gene that resulted in the conversion of a glutamic acid at codon 11 to a termination codon. The patient also inherited an XP-G allele from his mother that produces an unstable or poorly expressed message. The cause of the latter defect is still uncertain. In addition to these alterations, XP20BE cDNA contained an mRNA species with a large splicing defect that encompassed a deletion from exon 1 to exon 14. This splicing defect, however, appears to be a naturally occurring low-frequency event that results from abnormal splicing that occurs between certain conserved non-consensus splicing signals within the human XPG gene.

Alpha particle mutagenesis of human lymphoblastoid cell lines.

Despite being derived from the same donor, the human lymphoblastoid cell lines WTK1 and TK6 have markedly different responses to low LET radiation. We originally observed that WTK1 was more resistant to the cytotoxic effects of X-irradiation, but significantly more sensitive to mutation induction at both the TK and HPRT loci. In an effort to better understand these properties, we have examined the effects of alpha-particles on these cells. Relative to TK6, WTK1 has enhanced survival and mutation after both X-ray and alpha-particle exposure. While the HPRT locus was significantly more mutable in WTK1 as a function of alpha-particle versus X-ray dose, the TK locus was only slightly more sensitive to alpha-particle mutagenesis. In addition, the slowly growing TK mutants that constitute the majority of X-ray-induced TK mutants of TK6 were recovered in lower proportions following alpha-particle exposures. This is consistent with the further finding that in both cell lines, loss of heterozygosity occurred in a smaller fraction of alpha-induced TK mutants than X-ray-induced mutants. These results are consistent with our previous model suggesting that WTK1 has an error-prone repair pathway that is either missing or deficient in TK6, and further suggest that this pathway may be involved in the processing of alpha-particle-induced damage.

Genomic structure and chromosomal assignment of the mouse Ku70 gene.

DNA-dependent protein kinase (DNA-PK) consists of three polypeptide subunits: Ku70, Ku80, and the DNA-PK catalytic subunit (DNA-PKcs). Mammalian mutants deficient in either Ku80 or DNA-PKcs function have been shown to be lacking in DNA double-strand break repair and V(D)J recombination, respectively. The precise role of the Ku70 gene in this process has not yet been determined, in part because no cell lines, animals, or human diseases involved with deficiencies in this gene have yet been identified. Both the human and the mouse Ku70 cDNAs have been cloned, and the human gene has been mapped to chromosome 22q13. The original mouse cDNA clones, however, lacked a complete 5'-region, and none of the mammalian Ku70 genomic sequences have been characterized. This report contains an analysis of the 5'-region of the mouse cDNA sequence, a characterization of the mouse Ku70 genomic structure, and fluorescence in situ hybridization data that map the mouse gene to chromosome 15. The deduced amino acid sequence of the mouse gene consists of 608 amino acids compared to 609 for the human gene. The genomic sequence is 24 kb and consists of 13 exons, including an untranslated first exon. Sequences from the upstream region of exon 1 revealed four consensus GC box sequences and a strong transcription initiation site at a reasonable location. The assignment of the mouse Ku70 gene to chromosome 15 is consistent with the syntenic relationship of this gene in human (chromosome 22q13) and mouse and adds to the comparative mapping data for the genes involved in the SCID phenotype.

Molecular analysis and comparison of radiation-induced large deletions of the HPRT locus in primary human skin fibroblasts.

Genetic alterations in gamma-ray- and alpha-particle-induced HPRT mutants were examined by multiplex polymerase chain reaction (PCR) analysis. A total of 39-63% of gamma-ray-induced and 31-57% of alpha-particle-induced mutants had partial or total deletions of the HPRT gene. The proportion of these deletion events was dependent on radiation dose, and at the resolution limits employed there were no significant differences between the spectra induced by equitoxic doses of alpha particles (0.2-0.4 Gy) and gamma rays (3 Gy). The molecular nature of the deletions was analyzed by the use of sequence tagged site (STS) primers and PCR amplification as a "probe" for specific regions of the human X chromosome within the Xq26 region. These STSs were closely linked and spanned regions approximately 1.7 Mbp from the telomeric side and 1.7 Mbp from the centromeric side of the HPRT gene. These markers include: DXS53, 299R, DXS79, yH3L, 3/19, PR1, PR25, H2, yH3R, 1/44, 1/67, 1/1, DXS86, D8C6, DXS10 and DXS144. STS analyses indicated that the maximum size of total deletions in radiation-induced HPRT mutants can be greater than 2.7 Mbp and deletion size appears to be dependent on radiation dose. There were no apparent differences in the sizes of the deletions induced by alpha particles or gamma rays. On the other hand, deletions containing portions of the HPRT gene were observed to be 800 kbp or less, and the pattern of the partial deletion induced by alpha particles appeared to be different from that induced by gamma rays.

Non-radioactive mismatch analysis to detect small mutations in human hypoxanthine-guanine phosphoribosyl transferase cDNA.

We have combined a cDNA-driven PCR technique and a non-radioactive chemical-cleavage mismatch method, followed by a direct sequencing for detecting small mutations in the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene. HPRT cDNA was synthesized by RT-PCR from 1,000 wild-type or HPRT(-) mutant cells. Wild-type cDNA was hybridized with mutant cDNA to form heteroduplexes. The resultant mismatched bases were modified and cleaved by base-specific chemicals, followed by analysis by denaturing polyacrylamide gel electrophoresis. Cleaved fragments were detected without using radioactive materials. Finally, direct sequencing of the PCR products was performed with a focus on a small limited region indicated by the mismatch analysis (focused sequencing). In this study, three small mutations in exon-3 of HPRT cDNA were detected and characterized completely with this system. As compared with the radioactive method, this system was shown to be very simple and efficient.

Analysis of large X-ray-induced mutant populations by denaturing gradient gel electrophoresis.

To increase the precision by which predominant point mutations can be observed, hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient mutants selected en masse from large X-irradiated cultures of human lymphoblastoid cells (line TK6) were analyzed by denaturing gradient gel electrophoresis (DGGE). Four independent experiments yielded approximately 7 x 10(3) and 3.2 x 10(3) initial surviving 6-thioguanine-resistant (6-TGr) mutants in X-ray-treated and untreated cultures, respectively. The hprt exon 3 fragments were amplified from DNA extracted from these mixed 6-TGr cell populations by employing the polymerase chain reaction using modified T7 DNA polymerase. DGGE was used to separate the mutant sequences from the wild-type as mutant/wild-type heteroduplexes. The X-irradiated populations contained several mutant bands in the 104-bp low-melting region of exon 3 that were not observed in the untreated cultures. Two exon 3 specific mutations were observed in more than one treated culture and various tests for potential biases suggested that these were radiation-specific mutational hotspots. These two recurring mutations were specific 1-bp deletions in either a run of four T:A's (bp 294-297) or a run of 3 A:T's (bp 247-249). Several other "sporadic" signals observed in X-irradiated cultures were caused by small deletions ranging from 2 to 25 bp in length.

Differential effects induced by N-hydroxy-2-acetylaminofluorene and N-hydroxy-2-aminofluorene in DNA excision-repair-deficient Chinese hamster cells.

The direct-acting cytotoxic properties of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxy-2-aminofluorene (N-OH-AF) have been determined in repair-proficient (AA8-4) and repair-deficient (UV-5) Chinese hamster ovary cells. Cytotoxicity comparisons indicate that UV-5 cells are considerably more sensitive to exposure to N-OH-AAF than is the parental AA8-4 cell line, i.e., concentrations needed to obtain a D37 for survival of AA8-4 is greater than 5-fold higher than for UV-5. Mutation analysis at the HGPRT locus also indicates the increased sensitivity of UV-5 cells to N-OH-AAF as witnessed by an enhanced induction of 6-thioguanine-resistant colonies at equitoxic doses. Conversely, N-OH-AAF, did not induce a 'UV-mimetic' response when comparing genotoxicity between these two cell lines. Our data coupled with previously published model-building and adduct removal studies (Broyde and Hingerty, 1983; Fuchs and Daune, 1974; Grunberger and Weinstein, 1976; Yamasaki et al., 1977) suggest that the minor DNA adduct species, N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene, may be responsible for the hypermutagenicity witnessed in DNA excision-repair-deficient cells treated with N-OH-AAF.

2-Nitrofluoren-9-one: a unique mutagen formed in the photo-oxidation of 2-aminofluorene.

Exposure of solutions of 2-aminofluorene (2-AF, dissolved in dimethylsulfoxide) to near ultraviolet light (u.v.a. wavelengths of 320-400 nm) results in the formation of a variety of photo-products, several of which are direct-acting mutagens in the Ames/Salmonella standard-plate assay. Previously published results from our laboratory have described the chemical identification and kinetics of formation of two of these photo-induced mutagens, 2-nitrosofluorene and 2-nitrofluorene. In this report we present recent data concerning the isolation and chemical identification of another mutagenic photoproduct of u.v.a.-irradiated 2-AF, 2-nitrofluoren-9-one (2-NO2F-9-one). Data are also presented concerning the kinetics of phototransformation of 2-aminofluoren-9-one, an early-appearing and predominant photoproduct in u.v.a.-irradiated solutions of 2-AF, into 2-NO2F-9-one. It is well established that N-oxidation is a critical step in the biotransformations (i.e. enzymatic metabolism) of primary aromatic amines into proximate mutagens/carcinogens. In addition to u.v.a.-mediated N-oxidation of aromatic amines, selective ring photo-oxidation can also occur, resulting in, for example, the production of a carbonyl group at the 9-position of the fluorene molecule. The formation of mutagenic 2-NO2F-9-one in the photochemical oxidation of 2-AF appears to be unique to this process.

1-Nitropyrene: a mutagenic product induced by the action of near ultraviolet light on 1-aminopyrene.

A solution of 1-aminopyrene in dimethyl sulfoxide exposed to an artificial source of near ultraviolet light (600 kJ/m2) induced significant direct-acting mutagenicity in the Ames/Salmonella plating assay utilizing strain TA98. High-performance liquid chromatography of this solution resulted in a fraction that was mutagenic on TA98 but inactive on a nitroreductase-deficient strain of Salmonella (TA98NR). This observation suggested the presence of a nitro-containing compound. Mass spectral analysis confirmed that 1-nitropyrene was the active photoproduct in this fraction. These data implicate photochemical transformation of primary aromatic amines as an alternative mechanism by which nitroaromatic compounds can be formed in the environment.

Photochemical oxidation of 2-aminofluorene: correlation between the induction of direct-acting mutagenicity and the formation of nitro and nitroso aromatics.

The kinetics of near ultraviolet light-mediated phototransformation of 2-aminofluorene (2-AF) was studied using high performance liquid chromatography (HPLC) and the Ames/Salmonella mutagenicity bioassay. Employing tester strains TA98, TA1538, and the nitroreductase-deficient TA98NR without the addition of exogenous metabolic enzymes, we were able to detect and discriminate between the UVA exposure-dependent formation of two stable photoproducts, 2-nitrosofluorene (2-NOF) and 2-nitrofluorene (2-NO2F). Mutagenicity of irradiated 2-AF solutions (using dimethyl sulfoxide as a solvent) in the various tester strains indicates the rapid formation of the photo-labile 2-NOF, after which 2-NO2F accounts for the preponderance of mutagenic activity. Continued UVA irradiation (greater than 72 h at 6.8 J/m2/s) of 2-AF results in the formation of greater than 30 photoproducts resolvable on HPLC, several of which, in addition to 2-NOF and 2-NO2F, are mutagenic on Salmonella but are chemically undefined to date. Prolonged irradiation ultimately destroys the photo-induced mutagenicity of 2-AF. However, UVA-induced 2-AF photoproducts are stable for several weeks when stored in sealed vials in the dark. Light potentiated oxidation of aromatic amines constitutes an alternative mechanism for the transformation of aromatic amines into proximate mutagens/carcinogens.

Phototransformation of 2-aminofluorene into N-oxidized mutagens.

Exposure to sunlight or artificial sources of near u.v. light transforms 2-aminofluorene into a direct-acting mutagenic agent in the Ames/Salmonella histidine reversion bioassay. H.p.l.c. fractionation indicates that the majority of this mutagenic activity elutes as a single A254 absorbance peak. I.r. spectroscopy of the fractionated active fraction shows the presence of significant quantities of 2-nitrofluorene. The use of a nitroreductase deficient strain of Salmonella, coupled with t.l.c. analysis, however, also indicates the presence of a minor component whose mutagenic and t.l.c. properties are identical with 2-nitrosofluorene. These results implicate a specific mechanism by which aromatic amines can be photo-oxidized to potentially harmful genotoxic agents.

Genotoxicity induced in cultured Chinese hamster cells exposed to natural or synthetic crude oils and near ultraviolet light.

Cultured Chinese hamster ovary (CHO) cells were incubated with dilutions of natural or synthetic crude oils and subsequently exposed to near ultraviolet light (NUV). Although the magnitude of photo-induced cytotoxicity in CHO was essentially independent of the source of the oil (within a factor of two), two shale oils were exceptionally high in photo-induced mutagenic activity eliciting a response 10-12 times the observed natural background mutation frequency. Other shale oils, natural crude oils and a solvent refined coal medium distillate blend were weaker or negative in photo-induced mutagenic activity. Hydrotreatment of a photoactive shale oil, although eliminating the mutagenic potential, did not reduce the oil's cytotoxic potential.