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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 .

We present a simple approach that permits any circular plasmid, such as uncharacterized plasmids from diverse prokaryotes, to be established in Escherichia coli, thereby facilitating subsequent structural and functional studies. An in vitro transposition reaction is used to introduce a well-characterized replicon and selectable marker into purified plasmids, which are then used to transform E. coli. The approach was demonstrated using a small 3.4-kb archaeal plasmid and a large 60-kb uncharacterized plasmid from a Gram-negative bacterium. Replicon function in E. coli was tested for each plasmid, and direct sequencing of the large plasmid revealed similarity to restriction-modification systems.

Yersinia pestis, the causative agent of bubonic plague, emerged recently (

Rhizobium meliloti elicits the development of specialized organs on the plant roots called nodules. How nitrogen fixation genes are coordinately expressed specifically within the nodule is an important question in understanding the plant-microbe symbiosis. Homologs of nif genes have been identified in R. meliloti, but inactivation of these genes by transposon mutagenesis does not affect symbiosis. The fact that FixL* retains oxygen-regulated activity in vitro demonstrates that the amino-terminal membrane attachment domain is not essential for FixL function. The net effect, then, is an increase in the level of phospho-FixJ. The development of an oxygen-regulated in vitro transcription system is an important step in the study of FixJ action at target promoters. Two-component response regulators that function as transcription factors can be divided into three subclasses based on sequence similarity outside the universally conserved N-terminal phosphoryl acceptor domain. In contrast with many other systems, the phosphorylated forms of both the sensor kinase (FixL) and the response regulator (FixJ) are very stable, greatly facilitating many biochemical approaches. Signal transduction can be reconstituted in vitro, from stimulus to gene expression, with a minimum of water-soluble components. The study of FixL/FixJ may have important consequences in understanding other oxygen-regulated biological processes. Oxygen is an important regulator of many processes in bacteria. In addition to the intermolecular interactions between the kinase (transmitter) and phosphoryl acceptor (receiver) domains, other important intermolecular interactions may be important, such as monomer-dimer transitions.

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.

Comparative bacterial genomics shows that even different isolates of the same bacterial species can vary significantly in gene content. An effective means to survey differences across whole genomes would be highly advantageous for understanding this variation. Here we show that suppression subtractive hybridization (SSH) provides high, representative coverage of regions that differ between similar genomes. Using Helicobacter pylori strains 26695 and J99 as a model, SSH identified approximately 95% of the unique open reading frames in each strain, showing that the approach is effective. Furthermore, combining data from parallel SSH experiments using different restriction enzymes significantly increased coverage compared to using a single enzyme. These results suggest a powerful approach for assessing genome differences among closely related strains when one member of the group has been completely sequenced.

Yersinia pestis, the causative agent of bubonic plague, emerged recently (20000 years ago) as a clone of Yersinia pseudotuberculosis. There is scant evidence of genome diversity in Y. pestis, although it is possible to differentiate three biovars (antiqua, mediaevalis or orientalis) based on two biochemical tests. There are a few examples of restriction fragment length polymorphisms (RFLPs) within Y. pestis; however, their genetic basis is poorly understood. In this study, six difference regions (DFRs) were identified in Y. pestis, by using subtractive hybridization, which ranged from 4.6 to 19 kb in size. Four of the DFRs are flanked by insertion sequences, and their sequences show similarity to bacterial genes encoding proteins for flagellar synthesis, ABC transport, insect toxicity and bacteriophage functions. The presence or absence of these DFRs (termed the DFR profile) was demonstrated in 78 geographically diverse strains of Y. pestis. Significant genome plasticity was observed among these strains and suggests the acquisition and deletion of these DNA regions during the recent evolution of Y. pestis. Y. pestis biovar orientalis possesses DFR profiles that are different from antiqua and mediaevalis biovars, reflecting the recent origins of this biovar. Whereas some DFR profiles are specific for antiqua and mediaevalis, some DFR profiles are shared by both biovars. Furthermore, the progenitor of Y. pestis, Y. pseudotuberculosis (an enteric pathogen), possesses its own DFR profile. The DFR profiles detailed here demonstrate genome plasticity within Y. pestis, and they imply evolutionary relationships among the three biovars of Y. pestis, as well as between Y. pestis and Y. pseudotuberculosis.