A large motility operon, referred to as the flgB operon, was identified, characterized, and mapped at 310 to 320 kb on the linear chromosome of the spirochete Borrelia burgdorferi. This is the first report that a The bacterial motility apparatus is a highly organized but complicated structure composed of the flagellar filament, the hook-basal body complex, membrane-associated energy-transducing components, and proteins involved in export (1, 49, 69). Most of the flagellar genes of Escherichia coli and Salmonella typhimurium (1, 49), Bacillus subtilis (3,9,22,51,52,56,73), and Caulobacter crescentus (4, 19, 69) have been identified and sequenced. More than 50 genes are involved in flagellar structure and assembly, motility, and chemotaxis. These genes are organized into clusters and constitute functional operons. For example, in E. coli there are four motility and chemotaxis clusters (regions I, II, IIIa, and IIIb) consisting of 15 operons (49). These operons together comprise a large flagellar regulon. In contrast, the organization of the B. subtilis motilitychemotaxis genes is quite different (3,9,17,22,51,56,73). Although these genes are not clustered into four regions as is found in E. coli, the orders of certain flagellar genes in B. subtilis and E. coli are similar. The genes in B. subtilis are arranged such that several small flagellar clusters are involved in the last stage of flagellar assembly (17, 51, 52). In addition, there is a large (26-kb) fla/che locus containing more than 30 structural, motility, and chemotaxis genes (9). The genes encoding the proteins for early flagellar assembly are concentrated within this operon. In C. crescentus (4,19), the flagellar regulon is composed of more than 10 small operons with a gene order different from those in S. typhimurium, E. coli, and B. subtilis.The morphological pathway of flagellar assembly has been studied in detail for E. coli and S. typhimurium (1, 49) and C. crescentus (4, 69). Flagellar synthesis is highly coordinated, involving a specific order of gene expression which is achieved by a tightly controlled transcriptional cascade (1,4,19,60). In E. coli and S. typhimurium, the class 1 master proteins FlhD and FlhC are regulated under catabolite repression by glucose (49). Expression of both proteins results in the activation of all seven class 2 operons encoding components of the hook-basal body complex, the export apparatus, and the flagellum-specific sigma factor 28 (FliA) (36,44,49). The majority of the products encoded by class 2 genes are essential for full expression of class 3 genes.28 is utilized to initiate transcription of the seven class 3 operons. The class 3 gene products include the hook-associated proteins, flagellin, the motor proteins (MotA and MotB), and chemotaxis proteins. All class 3 genes and many of the class 2 genes, including fliA itself, are transcribed from 28 -like promoter elements (36,44,49). In B. subtilis, the genes corresponding to the class 2 and class 3 genes, including those from the large fla/che operon, are tra...

Background: Phi29 polymerase based amplification methods provides amplified DNA with minimal changes in sequence and relative abundance for many biomedical applications. RNA virus detection using microarrays, however, can present a challenge because phi29 DNA polymerase cannot amplify RNA nor small cDNA fragments (<2000 bases) obtained by reverse transcription of certain viral RNA genomes. Therefore, ligation of cDNA fragments is necessary prior phi29 polymerase based amplification. We adapted the QuantiTect Whole Transcriptome Kit (Qiagen) to our purposes and designated the method as Whole Transcriptome Amplification (WTA).

The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.

SummaryIdentification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole‐genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1–4 h. Using this method, we identified Monkeypox virus and drug‐resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner.

Physical maps of the chromosomes of the Lyme disease spirochaetes Borrelia garinii and Borrelia afielii have been elucidated for the enzymes Cspl, SgrAl, I-Ceul, Smal, Eagl, BssHII, MluI and Apal by two-dimensional pulsed-field gel electrophoresis techniques. The maps contain 42 sites for B. garinii and 32 for B. afielii. The mapping studies showed that the two chromosomes are linear DNA molecules of 953 and 948 kbp, respectively. A comparison of the physical maps of B. garinii and B. afielii and the published map of the other Lyme disease spirochaete, Borrelia burgdorferi [Davidson, B. E. , MacDougall, J. & Saint Girons, 1. (1992) J Bacteriol 174,3766-37741 revealed that the three chromosomes have f e w endonuclease sites in common, apart from a cluster in rrl (encoding 23s rRNA) and rrs (encoding 165 rRNA). Cloned borrelial genes were used as specific hybridization probes to construct genetic maps, using the physical maps as a basis. The resulting maps contain 41 genetic loci for B. burgdorferi, 39 for B. garinii, and 33 for B. afielii. In contrast to the physical maps, the three genetic maps are closely related, with no detectable differences in gene order along the entire length of the chromosome. It is concluded that the chromosomes of these three borrelial species have undergone no major rearrangements, deletions or insertions during their evolution from a common ancestor. Detailed mapping of the region of the B. garinii and B. afzelii chromosomes that encodes rRNA revealed that each chromosome contains one copy of rrs separated by 5 kbp from two copies each of rrl and rrf (encoding 55 rRNA). The gene order is rrs rrlA rrfA rrlB rrfB. B. burgdorferi is the only other member of the eubacteria for which this particular rRNA gene arrangement has been observed. A DNA length polymorphism in the region of the borrelial rRNA genes was shown to be due to the presence of 2.2 kbp more DNA between rrs and rrlA in B. garinii and B. afielii than in B. burgdorferi.