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Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification to sulfur compound oxidation; to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV); and to oxidize mineral electron donors. Recent analysis of its genome sequence also revealed the presence of genes encoding two [NiFe]hydrogenases, whose role in metabolism is unclear, as the sequenced strain does not appear to be able to grow on hydrogen as a sole electron donor under denitrifying conditions. In this study, we report the development of a genetic system for T. denitrificans , with which insertion mutations can be introduced by homologous recombination and complemented in trans . The antibiotic sensitivity of T. denitrificans was characterized, and a procedure for transformation with foreign DNA by electroporation was established. Insertion mutations were generated by in vitro transposition, the mutated genes were amplified by the PCR, and the amplicons were introduced into T. denitrificans by electroporation. The IncP plasmid pRR10 was found to be a useful vector for complementation. The effectiveness of the genetic system was demonstrated with the hynL gene, which encodes the large subunit of a [NiFe]hydrogenase. Interruption of hynL in a hynL ::kan mutant resulted in a 75% decrease in specific hydrogenase activity relative to the wild type, whereas complementation of the hynL mutation resulted in activity that was 50% greater than that of the wild type. The availability of a genetic system in T. denitrificans will facilitate our understanding of the genetics and biochemistry underlying its unusual metabolism.

The three species of the group 1 bacilli, Bacillus anthracis , B. cereus , and B. thuringiensis , are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis . Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis .

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.