A major obstacle to studying the functions of particular gene products in the mouse-tick infectious cycle of Borrelia burgdorferi has been an inability to knock out genes in pathogenic strains. Here, we investigated conditions for site-directed mutagenesis in B31 MI, the low-passage-number, infectious B. burgdorferi strain whose genome was sequenced. We inactivated several plasmid and chromosomal genes in B31 MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31 MI based on colony phenotype, growth rate, plasmid content, protein profile, and transformability. Significantly, several B31 MI clones that were not subjected to mutagenesis but that lacked particular plasmids also exhibited defects at various stages in the infectious cycle. Therefore, the high degree of clonal polymorphism within B31 MI complicates the assessment of the contributions of individual genes to the observed phenotypes of the mutants. Our results indicate that B31 MI is not an appropriate strain background for genetic studies in infectious B. burgdorferi, and a well-defined isogenic clone is a prerequisite for targeted mutagenesis. To this end, we derived several wild-type clones from B31 MI that were infectious for mice, and gene inactivation was successful in one of these clones. Due to the instability of the genome with in vitro propagation, careful monitoring of plasmid content of derived mutants and complementation of inactivated genes will be crucial components of genetic studies with this pathogen.Lyme disease is caused by Borrelia burgdorferi, a spirochete transmitted by ticks of the genus Ixodes and maintained within an enzootic cycle between the tick vector and mammalian hosts, most importantly small rodents (7,11,19). The clinical manifestations of this zoonosis can include a multisystem disorder affecting skin and joints and the nervous, lymphoreticular, and cardiovascular systems (39,40).The organization of the B. burgdorferi genome is unique among bacteria in that the genome is composed of a linear chromosome and a large number of linear and circular plasmids (8, 14). The complete genome sequence of an infectious B. burgdorferi isolate, the type strain B31, identified 21 linear and circular plasmids (8). In vitro propagation of B. burgdorferi can lead to plasmid loss and concurrent loss of infectivity for mice (3,21,22,30,33). Although increasing evidence suggests that certain Borrelia plasmids are important for infection in mice (18,25), this hypothesis has not been experimentally verified, and the roles of most plasmid-encoded genes in the infectious cycle are unknown. A number of plasmid-and chromosomally encoded genes have been inactivated in the highpassage-number, noninfectious clone B31-A (4,5,12,17,20,43,45,46), but gene inactivation in a low-passage-number, infectious strain background has not been reported.Here, we investigate conditions for site-directed mutagenesis in B31 MI, the low-passage-number, inf...

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There is now substantial evidence that Borrelia burgdorferi , the Lyme disease spirochete, undergoes major alterations in antigenic composition as it cycles between its arthropod and mammalian hosts. In this report, we cultivated B. burgdorferi 297 within dialysis membrane chambers implanted into the peritoneal cavities of rats to induce antigenic changes similar to those which occur during mammalian infection. Chamber-grown spirochetes, which remained fully virulent, did not express either outer surface protein A or Lp6.6, lipoproteins known to be downregulated after mammalian infection. However, they did, express p21, a well characterized outer surface protein E homologue, which is selectively expressed during infection. SDS-PAGE, two-dimensional gel electrophoresis, and immunoblot analysis revealed that chamber-grown borreliae also expressed uncharacterized proteins not expressed by in vitro-cultivated spirochetes; reactivity with sera from mice chronically infected with B. burgdorferi 297 confirmed that many of these novel proteins are selectively expressed during experimental murine infection. Finally, we used differential display RT-PCR to identify transcripts of other differentially expressed B. burgdorferi genes. One gene ( 2.9-7lpB ) identified with this technique belongs to a family of genes located on homologous 32-and 18-kb circular plasmids. The lipoprotein encoded by 2.9-7lpB was shown to be selectively expressed by chambergrown spirochetes and by spirochetes during experimental infection. Cultivation of B. burgdorferi in rat peritoneal implants represents a novel system for studying Lyme disease spirochetes in a mammalian host-adapted state. ( J. Clin. Invest. 1998. 101:2240-2250.) Key words: host-adapted • chamber implants • ticks • Borrelia burgdorferi • outer surface protein

Lyme disease is a tick-borne infection that can lead to chronic, debilitating problems if not recognized or treated appropriately. Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature by a complex enzootic cycle involving Ixodes ticks and mammalian hosts. Many previous studies support the notion that B. burgdorferi differentially expresses numerous genes and proteins to help it adapt to growth in the mammalian host. In this regard, several studies have utilized a dialysis membrane chamber (DMC) cultivation system to generate "mammalian host-adapted" spirochetes for the identification of genes selectively expressed during mammalian infection. Here, we have exploited the DMC cultivation system in conjunction with microarray technology to examine the global changes in gene expression that occur in the mammalian host. To identify genes regulated by only mammal-specific signals and not by temperature, borrelial microarrays were hybridized with cDNA generated either from organisms temperature shifted in vitro from 23°C to 37°C or from organisms cultivated by using the DMC model system. Statistical analyses of the combined data sets revealed that 125 genes were expressed at significantly different levels in the mammalian host, with almost equivalent numbers of genes being up-or down-regulated by B. burgdorferi within DMCs compared to those undergoing temperature shift. Interestingly, during DMC cultivation, the vast majority of genes identified on the plasmids were down-regulated (79%), while the differentially expressed chromosomal genes were almost entirely upregulated (93%). Global analysis of the upstream promoter regions of differentially expressed genes revealed that several share a common motif that may be important in transcriptional regulation during mammalian infection. Among genes with known or putative functions, the cell envelope category, which includes outer membrane proteins, was found to contain the most differentially expressed genes. The combined findings have generated a subset of genes that can now be further characterized to help define their role or roles with regard to B. burgdorferi virulence and Lyme disease pathogenesis.Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common arthropod-borne infection in the United States (39). Undiagnosed infection with B. burgdorferi often results in chronic disease, which can lead to sequelae such as carditis, arthritis, and neuritis (53). In nature, B. burgdorferi is maintained through a complex enzootic cycle involving ticks and mammalian hosts, typically small rodents (29). To perpetuate this enzootic cycle, B. burgdorferi must adapt physiologically to two dramatically different environments. Consistent with the adaptation process, several genes and the proteins they encode have been shown to be specifically up-regulated or down-regulated as this organism is transmitted from its tick vector to the mammalian host (1, 2, 18, 21, 23, 47). The bestcharacterized example of differential gene expression in B...

Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa ironregulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in this environment. We propose a model where P. aeruginosa lyses S. aureus and uses released iron for growth in low-iron environments.Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen commonly found in water and soil. P. aeruginosa causes a number of chronic and acute infections and is noted for its inherent resistance to many clinically relevant antibiotics. Two of the most common infections caused by P. aeruginosa are chronic colonization of the lungs of individuals with the genetic disease cystic fibrosis (CF) (19) and peritonitis in individuals undergoing continuous ambulatory peritoneal dialysis (CAPD) (25). The lungs of CF patients are commonly colonized before the age of 8, and most individuals maintain these infections throughout their lifetimes. High infection rates are also associated with CAPD, which is often used to treat end-stage renal disease.P. aeruginosa physiology and gene expression during in vivo growth is largely unknown. Using in vivo expression technology (IVET), Wang et al. identified 19 P. aeruginosa genes inducible during growth in a neutropenic mouse (40). Although that study identified several new genes important for virulence in P. aeruginosa, it did not provide a comprehensive analysis of in vivo gene expression. Two recent studies using Pasteurella multocida and Borrelia burgdorferi have provided a more comprehensive view of in vivo bacterial gene expression by using DNA microarrays (3, 4, 33). These studies illustrate that a significant number of genes (2 to 8% of all the genes in the genomes) are differentially regulated in vivo, suggesting that the in vivo environment is distinct from normal in vitro culture conditions.Although evaluation of the transcriptomes of in vivo-grown bacteria provides a snapshot of transcription under monoculture growth conditions, it is clear that many in...