Summary The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile.

Genetically distinct isolates of Coxiella burnetii, the cause of human Q fever, display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting that isolates have distinct virulence characteristics. To provide a more-complete understanding of C. burnetii's genetic diversity, evolution, and pathogenic potential, we deciphered the whole-genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. Cross-genome comparisons that included the previously sequenced Nine Mile (NM) reference isolate (RSA493) revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates are consistent with genome structures of other bacterial pathogens that have recently emerged from nonpathogens with expanded niches. The observation that the attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests that this isolate's lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages.

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB r , a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A 1 . The utility of the coumermycin-resistant gyrB r gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.Borrelia burgdorferi, the spirochetal agent of Lyme disease (4), is maintained in nature by an infectious cycle involving tick vectors and small rodent hosts (13). The genome sequence of B. burgdorferi has provided a wealth of data about the genetic composition of this bacterium (7). However, relatively little is known about the function of most of the deduced proteins encoded by the genome. For example, 41% of the chromosomal open reading frames and 84% of plasmid open reading frames are either homologs to hypothetical proteins in other bacteria or have no match in databases (7). Although many B. burgdorferi gene products have been identified and characterized on the basis of antigenicity, abundance, membrane location, or pattern of synthesis, the functions of most of these proteins are unknown (5,12,20,31,32,36). As a consequence, relatively little is known about the molecular mechanisms mediating B. burgdorferi variation and adaptation and the roles of these processes in the infectious cycle.Identification of genes that encode virulence factors or participate in the transmission cycle of B. burgdorferi is hindered because this pathogen differs considerably from bacteria with well-developed genetic systems. These differences include an atypical outer membrane composition, unique genome structure with an undefined mechanism of replication, and complex, stringent in vitro growth requirements. One major factor that has impeded genetic studies in B. burgdorferi is the lack of an exogenous selectable marker. The only available selectable marker was derived by mutating the B. burgdorferi gene for the B subunit of DNA gyrase (gyrB), yielding a derivative gene (gyrB r ) whose product confers resistance to the antibiotic coumermycin A 1 (26). When the gyrB r gene is used for gene inactivation by allelic exchange or integration into the genome, the most common outcome (Ͼ99.5%) is recombination with the endogenous chr...

Chlamydia trachomatis possesses a cryptic 7.5-kb plasmid of unknown function. Here, we describe a comprehensive molecular and biological characterization of the naturally occurring plasmidless human C. trachomatis strain L2(25667R). We found that despite minimal chromosomal polymorphisms, the LGV strain L2(25667R) was indistinguishable from plasmid-positive strain L2(434) with regard to its in vitro infectivity characteristics such as growth kinetics, plaquing efficiency, and plaque size. The only in vitro phenotypic differences between L2(434) and L2(25667R) were the accumulation of glycogen granules in the inclusion matrix and the lack of the typical intrainclusion Brownian-like movement characteristic of C. trachomatis strains. Conversely, we observed a marked difference between the two strains in their abilities to colonize and infect the female mouse genital tract. The 50% infective dose of plasmidless strain L2(25667R) was 400-fold greater (4 ؋ 10 6 inclusion-forming units [IFU]) than that of plasmid-bearing strain L2(434) (1 ؋ 10 4 IFU). Transcriptome analysis of the two strains demonstrated a decrease in the transcript levels of a subset of chromosomal genes for strain L2(25667R). Among those genes was glgA, encoding glycogen synthase, a finding consistent with the failure of L2(25667R) to accumulate glycogen granules. These findings support a primary role for the plasmid in in vivo infectivity and suggest that virulence is controlled, at least in part, by the plasmid's ability to regulate the expression of chromosomal genes. Our findings have important implications in understanding a role for the plasmid in the pathogenesis of human infection and disease.