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Lyme disease in the United States is most often caused by Borrelia burgdorferi sensu stricto. After a tick bite, the patient may develop erythema migrans at that site.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infectingBorrelia burgdorferispirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derivedB. burgdorferisensu stricto (Bbss) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification ofBbssisolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectiousBbssisolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ∼800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm acrossBbssisolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Background Onychomycosis affects almost 6% of the world population. Topical azoles and systemic antifungal agents are of low efficacy and can have undesirable side effects. An effective, non-invasive therapy for onychomycosis is an unmet clinical need. Objective Determine the efficacy threshold of non-thermal atmospheric plasma (NTAP) to treat onychomycosis in an in vitro model. Methods A novel toe/nail-plate model using cadaver nails and agarose media inoculated with Candida albicans was exposed to a range of NTAP doses. Results Direct exposure of C albicans and Trichophyton mentagrophytes to 12 minutes of NTAP results in complete killing at doses of 39 and 15 kPulses, respectively. Onset of reduced viability of C albicans to NTAP treatment through the nail plate occurs at 64 kPulses with 10× and 100× reduction at 212 and 550 kPulses, respectively. Conclusions NTAP is an effective, non-invasive therapeutic approach to onychomycosis that should be evaluated in a clinical setting.

Objective: To assess the microbial growth in unfortified and fortified Holder pasteurized donor human milk (HPDHM) during 96 hours of refrigerated storage in a clinical setting. Methods: Thirty-six unfortified samples and 77 fortified samples of HPDHM were prepared in a neonatal intensive care milk preparation room and stored in the NICU refrigerator at 4°C to simulate a real-life feeding environment. One milliliter aliquots were removed at 24-hour intervals and cultured in duplicate for bacterial growth on solid blood agar medium. Viable bacterial colonies were characterized using standard microbiological methods. Results: 96.5% of milk samples manipulated in a vertical laminar flow hood were negative for bacterial growth. In the remainder 3.5% of the samples, the maximum growth was 1 colony forming unit/0.1 ml plated. Higher colony counts were observed when the laminar hood was not used. In all cases, the colonies represented common skin bacteria and demonstrated an inconsistent and unsustained growth. Fortifier status and storage time were not significantly associated with increased bacterial growth (P > 0.05). Conclusions: Unfortified and fortified HPDHM remain largely free of bacterial growth for up to 96 hours of refrigerated storage in NICU settings. Sample handling techniques are important for preventing microbial contamination.

Lyme disease (LD) is an increasing public health problem. Current laboratory testing is insensitive in early infection, the stage at which appropriate treatment is most effective in preventing disease sequelae. The Lyme Disease Biobank (LDB) collects samples from individuals with symptoms consistent with early LD presenting with or without erythema migrans (EM) or an annular, expanding skin lesion and uninfected individuals from areas of endemicity. Samples were collected from 550 participants (298 cases and 252 controls) according to institutional review board-approved protocols and shipped to a centralized biorepository., Lyme disease (LD) is an increasing public health problem. Current laboratory testing is insensitive in early infection, the stage at which appropriate treatment is most effective in preventing disease sequelae. The Lyme Disease Biobank (LDB) collects samples from individuals with symptoms consistent with early LD presenting with or without erythema migrans (EM) or an annular, expanding skin lesion and uninfected individuals from areas of endemicity. Samples were collected from 550 participants (298 cases and 252 controls) according to institutional review board-approved protocols and shipped to a centralized biorepository. Testing was performed to confirm the presence of tick-borne pathogens by real-time PCR, and a subset of samples was tested for Borrelia burgdorferi by culture. Serology was performed on all samples using the CDC’s standard two-tiered testing algorithm (STTTA) for LD. LD diagnosis was supported by laboratory testing in 82 cases, including positive results by use of the STTTA, PCR, or culture or positive results by two enzyme-linked immunosorbent assays for cases presenting with EM lesion sizes of >5 cm. The remaining 216 cases had negative laboratory testing results. For the controls, 43 were positive by at least one of the tiers and 6 were positive by use of the STTTA. The results obtained with this collection highlight and reinforce the known limitations of serologic testing in early LD, with only 29% of individuals presenting with EM lesion sizes of >5 cm yielding a positive result using the STTTA. Aliquots of whole blood, serum, and urine from clinically characterized patients with and without LD are available to investigators in academia and industry for evaluation or development of novel diagnostic assays for LD, to continue to improve upon currently available methods.

Genetic diversity of Anaplasma phagocytophilum was assessed in specimens from 16 infected patients and 16 infected Ixodes scapularis ticks. A region immediately downstream of the 16S rRNA gene, which included the gene encoding SdhC, was sequenced. For the A. phagocytophilum strains from patients no sequence differences were detected in this region. In contrast, significantly fewer ticks had a sequence encoding SdhC that was identical to that of the human strains (11/16 vs. 16/16, p = 0.04). This variation is consistent with the premise that not all A. phagocytophilum strains present in nature are able to cause clinical illness in humans. A strain referred to as A. phagocytophilumVariant-1 that is regarded as non-pathogenic for humans was previously described using a different typing method. Data from the current study suggest that both typing methods are identifying the same non-pathogenic strains.

Endothelial glycocalyx (EG) is disintegrated during sepsis. We have previously shown that this occurs very early in the course of sepsis and its prevention improves the survival of mice with sepsis. Here, we sought to investigate the possibility of pharmacologically accelerating the restoration of disintegrated EG in sepsis. We used a soilage injection model to induce polymicrobial sepsis in C57/BL6 mice and measured total body EG. En face aortic preparations were used for staining of markers of EG and atomic force microscopy was used to measure EG in vitro. In vitro studies were conducted in cultured endothelial cells either exposed to a lipopolysaccharide or enzymatically denuded of EG. Sulodexide (SDX), a heparin sulfate-like compound resistant to degradation by heparanase, accelerated EG regeneration in vitro and in vivo. The total volume of EG was drastically reduced in septic mice. Administration of SDX produced a dramatic acceleration of EG restoration. This effect, unrelated to any SDX-induced differences in microbial burden, was associated with better control of vascular permeability. Notably, SDX demonstrated not only a remarkable capacity for EG regeneration in vitro and in vivo but was also associated with improved animal survival, even when instituted 2 hours after induction of severe sepsis. In conclusion, 1) EG is disintegrated in sepsis, the event which contributes to high animal mortality; 2) pharmacologic acceleration of EG restoration can be achieved using SDX; and 3) SDX reduces vascular permeability, which is elevated in septic mice, and improves animal survival.

Lyme disease is transmitted by the bite of certain Ixodes ticks, which can also transmit Anaplasma phagocytophilum , the cause of human granulocytic anaplasmosis (HGA). Although culture can be used to identify patients infected with A. phagocytophilum and is the microbiologic gold standard, few studies have evaluated culture-confirmed patients with HGA. We conducted a prospective study in which blood culture was used to detect HGA infection in patients with a compatible clinical illness. Early Lyme disease was defined by the presence of erythema migrans. The epidemiologic, clinical, and laboratory features of 44 patients with culture-confirmed HGA were compared with those of a convenience sample of 62 patients with early Lyme disease. Coinfected patients were excluded. Patients with HGA had more symptoms ( P = 0.003) and had a higher body temperature on presentation ( P < 0.001) than patients with early Lyme disease. HGA patients were also more likely to have a headache, dizziness, myalgias, abdominal pain, anorexia, leukopenia, lymphopenia, thrombocytopenia, or elevated liver enzymes. A direct correlation between the number of symptoms and the duration of illness at time of presentation (rho = 0.389, P = 0.009) was observed for HGA patients but not for patients with Lyme disease. In conclusion, although there are overlapping features, culture-confirmed HGA is a more severe illness than early Lyme disease.

Bloodstream invasion is an important event in the pathogenesis of the more serious manifestations of Lyme disease. The number of spirochetes in the blood of infected patients, however, has not been determined, and, therefore, it is unknown whether the number of spirochetes can be correlated with particular clinical or laboratory features. This study was designed to measure the level of Borrelia burgdorferi in the plasma of Lyme disease patients and correlate these levels with selected clinical and laboratory findings. Nested and quantitative polymerase chain reaction (qPCR) was employed to detect cell-associated flaB gene DNA in the plasma of untreated early Lyme disease patients with erythema migrans (EM). Twenty-nine (45.3%) of 64 patients had evidence of B. burgdorferi in their plasma by at least one of the PCR methods. For the 22 qPCR-positive patients, the mean number of flaB gene copies per mL of plasma was 4,660, with a range of 414 to 56,000. The number of flaB gene copies did not significantly correlate with any of the clinical, demographic, or laboratory variables assessed. For reasons discussed, we suggest caution in extrapolating an estimate of the number of viable Borrelia in plasma from the observed number of flaB copies.

Bacterial translation initiation factor 3 (IF3) is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal domain (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain, but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect. Here, the Y75N substitution mutation in the NTD is examined in vitro and in vivo. IF3(Y75N) protein binds 30S subunits normally, but is defective in start-codon discrimination, inhibition of initiation on leaderless mRNA, and initiator-tRNA selection, thereby establishing a direct role for the IF3 NTD in these initiation processes. A model illustrating how IF3 modulates an inherent function of the 30S subunit is discussed.

Detection of antibody to C6, a 26–amino acid peptide that reproduces the sequence of the sixth invariable region (IR6) within the central domain of the VlsE protein of Borrelia burgdorferi sensu lato, is currently used for the serologic diagnosis of Lyme disease [1–4]. A potential concern with a test based on a single peptide is the possibility of sequence variation and a consequent lack of antigenic cross-reactivity among pathogenic strains of B. burgdorferi sensu lato, leading to reduced diagnostic sensitivity in some infected patients. B. burgdorferi sensu stricto (hereafter referred to as B. burgdorferi) isolates can be categorized into 3 distinct ribosomal spacer restriction fragment length polymorphism genotypes (RSTs) on the basis of analysis of the 16S-23S ribosomal DNA (rDNA) spacer region [5, 6]. North American isolates of B. burgdorferi can also be differentiated into at least 16 major genetic groups on the basis of variability in the genetic sequence encoding outer surface protein C (OspC) [7, 8]. Studies employing these methods have demonstrated that pathogenicity is dependent at least in part on the genotype of B. burgdorferi causing the infection; for example, evidence indicates that RST1 and RST2 are more likely to be associated with hematogenous dissemination in humans than is RST3 [6]. Although serologic testing of patients with erythema migrans is not routinely recommended for the management of these patients, serum samples from such patients are often used to validate or compare serologic assays, because this is the most common manifestation of Lyme disease [9], and it is the only manifestation for which the diagnosis can frequently be confirmed by the microbiologic gold standard of recovering B. burgdorferi on culture [10]. In a previous study [11], we demonstrated the comparable sensitivity of a C6 assay in 79 patients from North America with erythema migrans, irrespective of the RST genotype that caused the infection. The current study expands on the previous study by increasing the number of patient serum samples evaluated and by also considering the ospC genotype of the infecting strain of B. burgdorferi. In addition, the effect of the genotype of B. burgdorferi on the sensitivity of 2-tier testing was evaluated.

RecA is a key protein linking genetic recombination to DNA replication and repair in bacteria. Previous functional characterization of Borrelia burgdorferi RecA indicated that the protein is mainly involved in genetic recombination rather than DNA repair. Genetic recombination may play a role in B. burgdorferi persistence by generation of antigenic variation. We report here the isolation of a recA null mutant in an infectious B. burgdorferi strain. Comparison of the in vitro growth characteristics of the mutant with those of the wild-type strain under various conditions showed no significant differences. While the RecA mutant was moderately more sensitive to UV irradiation and mitomycin C than the wild-type strain, the lack of RecA abolished allelic exchange in the mutant. Absence of RecA did not affect the ability of the mutant to infect mice. However, the RecA mutant was attenuated for joint infection in competitive-infection assays with the wild-type strain. vlsE sequence variation in mice was observed in both wild-type and RecA mutant spirochetes, indicating that the mechanism of antigenic variation is not homologous genetic recombination.

The outer membrane of Borrelia burgdorferi , the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% α-helix with little β-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.

Human granulocytic anaplasmosis (HGA) is a potentially fatal tick-borne infection caused by Anaplasma phagocytophilum. Treatment options are limited for this entity, with doxycycline being the drug of choice. Certain fluoroquinolones such as levofloxacin are active against A. phagocytophilum in vitro. We report a hospitalized patient with HGA who improved coincident with a 13-day course of levofloxacin therapy, but clinically and microbiologically relapsed 15 days after completion of treatment. Relapse of infection after levofloxacin therapy was reproduced in a severe combined immune-deficient (SCID) mouse infection model. Quinolone therapy should not be considered curative of HGA.

Borrelia burgdorferi , the etiologic agent of Lyme disease, is genetically heterogeneous. Previous studies have shown a significant association between the frequency of hematogenous dissemination in Lyme disease patients and the genotype of the infecting B. burgdorferi strain. Comparative transcriptional profiling of two representative clinical isolates with distinct genotypes (BL206 and B356) was undertaken. A total of 78 open reading frames (ORFs) had expression levels that differed significantly between the two isolates. A number of genes with potential involvement in nutrient uptake (BB0603, BBA74, BB0329, BB0330, and BBB29) have significantly higher expression levels in isolate B356. Moreover, nearly 25% of the differentially expressed genes are predicted to be localized on the cell surface, implying that these two isolates have cell surface properties that differ considerably. One of these genes, BBA74, encodes a protein of 257 amino acid residues that has been shown to possess porin activity. BBA74 transcript level was >20-fold higher in B356 than in BL206, and strain B356 contained three- to fivefold more BBA74 protein. BBA74 was disrupted by the insertion of a kanamycin resistance cassette into the coding region. The growth rates of both wild-type and mutant strains were essentially identical, and cultures reached the same final cell densities. However, the mutant strains consistently showed prolonged lags of 2 to 5 days prior to the induction of log-phase growth compared to wild-type strains. It is tempting to speculate that the absence of BBA74 interferes with the enhanced nutrient uptake that may be required for the entry of cells into log-phase growth. These studies demonstrate the value of comparative transcriptional profiling for identifying differences in the transcriptomes of B. burgdorferi clinical isolates that may provide clues to pathogenesis. The 78 ORFs identified here are a good starting point for the investigation of factors involved in the hematogenous dissemination of B. burgdorferi .

Lyme disease is the most common tickborne disease in the United States [1]. Although the majority of cases are reported from the mid-Atlantic, northeastern, midwestern, and far western regions of the country, several hundreds of cases annually are reported from the southeast and south-central United States [1, 2]. Many of these cases are associated with an erythema migrans (EM)–like rash [3–6]. This skin lesion has followed the bite of Amblyomma americanum, a tick species found throughout the southeast and south-central United States [4, 7–9], and is alternatively referred to as southern tick–associated rash illness or Masters disease. A. americanum ticks are not infected with Borrelia burgdorferi but may be infected with a borrelial species different from both B. burgdorferi and the other Borrelia genospecies recognized to cause Lyme disease in Eurasia [10–19]. Barbour et al. [11] proposed the name Borrelia lonestari species novum. Until recently, it remained unclear whether A. americanum could transmit B. lonestari to humans or whether B. lonestari was the cause of (or even associated with) any clinical illness. In 2001, James et al. [18] documented the first such case. This patient developed an EM-like rash following the bite of an A. americanum tick during a trip to Maryland and North Carolina. James et al. [18] demonstrated by PCR amplification techniques that both the tick and the patient were infected with B. lonestari or a closely related bacterium. An important question is whether B. lonestari is the cause of the many cases reported as Lyme disease in southern and other regions of the United States where A. americanum ticks bite humans. In this article, we report results of a microbiologic evaluation of patients from Missouri with EM-like skin lesions and compare these results with those for patients from New York State with EM who were similarly evaluated during the same period.

Lyme disease, the most commonly reported tick-borne infection in North America, is caused by infection with the spirochete Borrelia burgdorferi. Although an accurate clinical diagnosis can often be made based on the presence of erythema migrans, in research studies microbiologic or molecular microbiologic confirmation of the diagnosis may be required. In this study, we evaluated the sensitivity of 5 direct diagnostic methods (culture and nested polymerase chain reaction [PCR] of a 2-mm skin biopsy specimen, nested PCR and quantitative PCR (qPCR) performed on the same 1-mL aliquot of plasma and a novel qPCR-blood culture method) in 66 untreated adult patients with erythema migrans. Results of one or more of these tests were positive in 93.9% of the patients. Culture was more sensitive than PCR for both skin and blood, but the difference was only statistically significant for blood samples (P

Approximately 45% of untreated United States patients with early Lyme disease associated with erythema migrans have a positive blood culture based on microscopic detection of Borrelia burgdorferi in Barbour-Stoenner-Kelly medium after 2 to 12 weeks of incubation. In this study we demonstrate that the yield of blood cultures can be significantly increased to 70.8% by the use of a combined culture-quantitative PCR technique and that among those patients found to have a positive blood culture, positivity was detected in over 90% within just 7 days of incubation. Patients with multiple erythema migrans were almost uniformly culture positive by this technique.

Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between reservoirs and hosts by ticks of the family Ixodidae. Infection with B. burgdorferi in humans causes Lyme disease or Lyme borreliosis. Currently, 20 Lyme disease-associated Borrelia species and more than 20 relapsing fever-associated Borrelia species have been described. Identification and differentiation of different Borrelia species and strains is largely dependent on analyses of their genetic characteristics. A variety of molecular techniques have been described for Borrelia isolate speciation, molecular epidemiology, and pathogenicity studies. In this unit, we focus on three basic protocols, PCR-RFLP-based typing of the rrs-rrlA and rrfA-rrlB ribosomal spacer, ospC typing, and MLST. These protocols can be employed alone or in combination for characterization of B. burgdorferi isolates or directly on uncultivated organisms in ticks, mammalian host reservoirs, and human clinical specimens.

Recently, a number of refinements in diagnostic modalities for detection of Borrelia burgdorferi infection have been developed. These include large-volume blood cultures, quantitative polymerase chain reaction (PCR) techniques, and 2-stage serologic testing. In the present study, we compared 6 diagnostic modalities in 47 adult patients who had a clinical diagnosis of erythema migrans. Quantitative PCR on skin biopsy-derived material was the most sensitive diagnostic method (80.9%), followed by 2-stage serologic testing of convalescent-phase samples (66.0%), conventional nested PCR (63.8%), skin culture (51.1%), blood culture (44.7%), and serologic testing of acute-phase samples (40.4%). Results of all assays were negative for 3 patients (6.4%). We conclude that the clinical diagnosis of erythema migrans is highly accurate in an area where B. burgdorferi is endemic if it is made by experienced health care personnel, but some patients with this diagnosis may not have B. burgdorferi infection. No single diagnostic modality is suitable for detection of B. burgdorferi in every patient with erythema migrans.

Genetic diversity among Borrelia burgdorferi isolates recovered from the skin of Lyme disease patients was assessed by ribosomal DNA (rDNA) spacer restriction fragment length polymorphism analysis, genomic restriction site polymorphism analysis, and plasmid content analysis. There was a significant association between the three rDNA spacer types, the six pulsed-field gel types, and plasmid content ( P < 0.001). The association between distinct chromosomal and plasmid markers implies a clonal origin for each genotype.

To investigate whether genetic diversity of Borrelia burgdorferi sensu stricto may affect the occurrence of hematogenous dissemination, 104 untreated adults with erythema migrans from a Lyme disease diagnostic center in Westchester County, New York, were studied. Cultured skin isolates were classified into 3 groups by a polymerase chain reaction amplification and restriction fragment length polymorphism (RFLP) method. A highly significant association between infecting RFLP type in skin and the presence of spirochetemia was found (P

Human granulocytic ehrlichiosis was first described in the United States, in the northern Midwest, in 1994.1 Human granulocytic ehrlichiosis is caused by an organism, still referred to as the agent of human granulocytic ehrlichiosis, that is similar to two animal pathogens, Ehrlichia phagocytophila and E. equi. 2–4 Transmission of human granulocytic ehrlichiosis occurs through the bites of ixodes ticks, which are the arthropod vectors for Borrelia burgdorferi and Babesia microti. 5,6 Human granulocytic ehrlichiosis is an acute, febrile, nonspecific illness that may be severe enough to cause hospitalization and even death, particularly in the elderly.1,7,8 We describe a . . .

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.

Background Erythema migrans is the most common manifestation of Lyme disease. Recurrences are not uncommon, and although they are usually attributed to reinfection rather than relapse of the original infection, this remains somewhat controversial. We used molecular typing of Borrelia burgdorferi isolates obtained from patients with culture-confirmed episodes of erythema migrans to distinguish between relapse and reinfection. Methods We determined the genotype of the gene encoding outer-surface protein C (ospC) of B. burgdorferi strains detected in cultures of skin or blood specimens obtained from patients with consecutive episodes of erythema migrans. After polymerase-chainreaction amplification, ospC genotyping was performed by means of reverse lineblot analysis or DNA sequencing of the nearly full-length gene. Most strains were further analyzed by determining the genotype according to the 16S–23S ribosomal RNA intergenic spacer type, multilocus sequence typing, or both. Patients received standard courses of antibiotics for erythema migrans. Results B. burgdorferi isolates obtained from 17 patients who received a diagnosis of erythema migrans between 1991 and 2011 and who had 22 paired episodes of this lesion (initial and second episodes) were available for testing. The ospC genotype was found to be different at each initial and second episode. Apparently identical genotypes were identified on more than one occasion in only one patient, at the first and third episodes, 5 years apart, but different genotypes were identified at the second and fourth episodes. Conclusions None of the 22 paired consecutive episodes of erythema migrans were associated with the same strain of B. burgdorferi on culture. Our data show that repeat episodes of erythema migrans in appropriately treated patients were due to reinfection and not relapse. (Funded by the National Institutes of Health and the William and Sylvia Silberstein Foundation.)

The etiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, has been isolated from many biologic sources in North America and Eurasia, and isolates have been divided into three distinct genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). In order to explore the possible association of genospecies with disease manifestation, 60 isolates of B. burgdorferi sensu lato were subjected to 5S rDNA-linked restriction fragment length polymorphism (RFLP) analysis. The results confirmed earlier studies which indicated that virtually all North American isolates are B. burgdorferi sensu stricto, whereas Eurasian strains fall into all three genospecies. Thirty-five isolates were further characterized by PCR amplification of a region of the 16S-23S rDNA spacer and HinfI digestion of the products. This method resulted in the subdivision of B. burgdorferi sensu stricto into two distinct PCR-RFLP types. In contrast, B. garinii isolates all displayed an identical pattern. Additionally, a number of previously unclassified North American isolates (25015, DN127, 19857, 24330) showed distinctively different PCR-RFLP patterns. The application of this method for the typing of uncultured B. burgdorferi directly in biologic samples was demonstrated by analysis of several field-collected Ixodes scapularis tick specimens. The described PCR-RFLP technique should allow for the direct and rapid molecular typing of B. burgdorferi-containing samples and facilitate studies of the relationship between spirochete genotype and clinical disease.

The capacity for Borrelia burgdorferi to cause disseminated infection in humans or mice is associated with the genotype of the infecting strain. The cytokine profiles elicited by B. burgdorferi clinical isolates of different genotype (ribosomal spacer type) groups were assessed in a human PBMC co-incubation model. RST1 isolates, which are more frequently associated with disseminated Lyme disease in humans and mice, induced significantly higher levels of IFN-α and IFN-λ1/IL29 relative to RST3 isolates, which are less frequently associated with disseminated infection. No differences in the protein concentrations of IFN-γ, IL-1β, IL-6, IL-8, IL-10 or TNF-α were observed between isolates of differing genotype. The ability of B. burgdorferi to induce type I and type III IFNs was completely dependent on the presence of linear plasmid (lp) 36. An lp36-deficient B. burgdorferi mutant adhered to, and was internalized by, PBMCs and specific dendritic cell (DC) subsets less efficiently than its isogenic B31 parent strain. The association defect with mDC1s and pDCs could be restored by complementation of the mutant with the complete lp36. The RST1 clinical isolates studied were found to contain a 2.5-kB region, located in the distal one-third of lp36, which was not present in any of the RST3 isolates tested. This divergent region of lp36 may encode one or more factors required for optimal spirochetal recognition and the production of type I and type III IFNs by human DCs, thus suggesting a potential role for DCs in the pathogenesis of B. burgdorferi infection.

An 11,955-bp region of the Borrelia burgdorferi chromosome containing all the genes encoding ribosomal RNA (rRNA) has been sequenced. The region contains a single gene encoding 16S rRNA and two genes encoding the 23S and 5S rRNAs. The sizes of the 16S, 23S and 5S rRNAs encoded by these genes are 1537, 2926 and 112 nucleotides, respectively. In addition, the genes encoding tRNA(Ala) and tRNA(Ile) are located in the intergenic spacer between the 16S and 23S rDNAs. The tDNAs do not encode the common CCA 3' end which presumably must be added posttranscriptionally. All the genes are present in the same orientation, except for that encoding tRNA(Ile), which is transcribed from the opposite strand. The latter implies that the rDNAs are not transcribed as a single unit. The location of putative promoters and termination signals in the sequence suggest that the 16S rRNA and tRNA(Ala) are transcribed as a single unit, tRNA(Ile) is produced as an individual transcript and the 23S and 5S rDNAs are co-transcribed. Several of the features of this rDNA organization are unique, not having been described previously in any other eubacteria.

Translation initiation factor IF3 plays a crucial role in initiation of protein synthesis in bacteria. In order to elucidate the IF3 structural elements required for these functions, the evolutionary conservation of IF3 and its gene, infC, was investigated. Homologous infC sequences from Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens and Proteus vulgaris were amplified by the polymerase chain reaction and sequenced. Analysis of these sequences, as well as that from Bacillus stearothermophilus, revealed several regions (e.g. residues 62-73 and 173-177) of absolute sequence conservation, suggesting an important role for these regions in IF3 function.

Lyme disease, the most common arthropod-borne infection in the United States, is caused by the spirochete Borrelia burgdorferi (87). In nature, B. burgdorferi has an obligate biannual enzootic cycle involving small mammalian host reservoirs, typically Peromyscus leucopus, and an Ixodes tick vector (51, 96). To successfully complete this life cycle, B. burgdorferi must adapt to and propagate within two markedly different physiologic milieus (67, 75, 96). A number of investigators have reported that manipulation of parameters, such as temperature, pH, DNA supercoiling, cell density, and partial O2 pressure, during in vitro cultivation can trigger changes in gene and protein expression resembling those that occur when spirochetes adapt to their mammalian host (4, 20, 21, 44, 45, 65, 74, 77, 78, 89, 102). It also is now evident, however, that spirochetes must be exposed to as yet unidentified mammalian host-specific signals to induce the full range of transcriptional and translational changes that occur during infection (1, 13, 17, 19, 39, 97). Gene regulation during the tick phase of the enzootic cycle has come to be recognized as equally important to the spirochete's survival strategy (24, 29, 32, 53, 67, 73, 74, 91, 92, 96). In this regard, recent evidence obtained from expression profiling of p66 and ospD points to the existence of arthropod-specific signals that modulate B. burgdorferi gene expression at various times during the tick phases of the cycle (24, 91). Studies of differential gene expression in B. burgdorferi have given rise to several regulatory paradigms (90). The two most extensively investigated are the reciprocal regulation of outer surface protein A (ospA) and ospC as spirochetes alternate between the arthropod vector and mammalian host (25, 29, 41, 61, 64, 68, 76, 77, 106) and the temperature- and blood meal-dependent induction of the ospE, ospF, and elp (erp) genes (3, 6, 28, 39, 58, 88). An important advance in our understanding of the molecular mechanisms underlying these paradigms was the discovery from the genomic sequence that Lyme disease spirochetes coordinate their physiologic adaptations and pathogenic programs with just three sigma factors: the housekeeping sigma factor, σ70 (RpoD), and the alternate sigma factors RpoN and RpoS (33). Seminal studies by Norgard and coworkers (42, 83, 103) demonstrated that expression of RpoS is regulated by RpoN in concert with the response regulator protein Rrp2. Expression of RpoS, in turn, is essential for the induction of the established virulence factors ospC, dbpBA, and bbk32 as well as numerous other genes thought to be required to establish mammalian infection (18, 19, 42, 59, 79, 80, 100). Analysis of rpoS and RpoS-dependent genes within infected ticks has demonstrated that induction of the RpoS regulon begins during the nymphal blood meal prior to spirochete transmission (i.e., the RpoS-ON state) (19, 35, 36, 39, 41, 76, 77). RpoS is also required for repression of ospA and other tick-phase genes (e.g., bba62 and lp6.6) in response to mammalian host-specific signals (17, 19) although it is not known if RpoS-dependent repression occurs during nymphal tick feeding or only after spirochetes have transited to their murine host. Because ospA is transcribed by σ70 (84), the repression mechanism most likely involves blockage of transcription by an RpoS-dependent trans-acting factor; we along with others have proposed that the poly(T) tract upstream of the ospA promoter also contributes to repression (13, 17, 19, 84). Downregulation of ospA may also be influenced by environmental factors, such as pH (102), and or DNA topology (4). Expression of genes downregulated by RpoS within the mammalian host is thought to resume once spirochetes are acquired by naive larvae (i.e., the RpoS-OFF state) (19). Although the presence of homologous upstream regions and highly similar expression profiles initially suggested that the ospE, ospF, and elp genes are controlled by a common regulatory mechanism (2, 28, 58, 89), expression of ospF has been shown to be RpoS dependent while the ospE and elp paralogs are transcribed by σ70 (17, 18, 26, 27). Promoter mapping studies revealed that sequences in the −10 regions of the ospE, ospF, and elp promoters play a critical role in determining recognition by σ70 or RpoS (27). Transcription of ospE and elp paralogs by σ70 would make the corresponding proteins available at points within the enzootic cycle when RpoS is not present. By analogy with regulatory mechanisms identified in other bacteria (14, 37), differential expression of σ70-dependent genes in B. burgdorferi presumably involves trans-acting factors that interact directly or indirectly with RNA polymerase to enhance or diminish the efficiency of gene transcription. Along these lines, Babb et al. (5) have reported that the Erp-binding factor, chromosomal (EbfC), a borrelial YbaB ortholog (55), binds to a region upstream of the ospE paralogs although it is unclear what, if any, effect EbfC binding has on transcription of these genes. BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function (81, 82). More recently, we demonstrated that recombinant BBA74 lacks the physical properties typical of porins and that the native protein is located in the periplasmic space of B. burgdorferi (63). Bioinformatics analysis, however, has been unable to elucidate a possible function for BBA74 as it has no known orthologs. A clue that BBA74 functions within the arthropod vector was provided by microarray studies of spirochetes cultivated within dialysis membrane chambers (DMCs), which showed that bba74 is downregulated in response to mammalian host-specific signals (13, 19). To extend these results, we performed a more thorough characterization of bba74 in comparison to the paradigmatic genes ospC, ospA, and ospE. These analyses revealed that bba74 is transcribed by σ70 exclusively during the larval and nymphal blood meals and that this novel expression pattern is a result of RpoS-independent and -dependent forms of repression that are induced by arthropod host- and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability of Borrelia to complete its infectious life cycle (8, 36, 91), the complex regulation of this gene is consistent with the notion that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. Our analysis of bba74 expression during the enzootic cycle also provided an opportunity to reexamine the ospA-ospC regulatory paradigm (75, 90, 96). Contrary to the prediction of strictly reciprocal expression of these genes and their corresponding proteins, spirochetes in the midguts of fed nymphs express large amounts of OspA as well as OspC. Our findings suggest that the heterogeneous expression of OspA and OspC by spirochete populations within fed nymphs results from the intricate sequence of transcriptional and translational changes that ensue as spirochetes transition from the RpoS-OFF to RpoS-ON state.

infC, the gene encoding translation initiation factor IF3 in Escherichia coli, can be transcribed from three promoters. Two of these promoters, PI1 and PI2, are located in the upstream thrS sequence which codes for threonyl-tRNA synthetase. Previous studies had shown that PI2 was the major promoter for infC. In the present study, the extent of transcription from PI1 and/or PI2 at a variety of steady-state growth rates was analyzed by promoter fusion studies. PI2 was the more active promoter (two- to threefold stronger than PI1) at all growth rates tested. A fusion plasmid containing both PI1 and PI2 exhibited a transcription level approximately equal to the sum of those observed with the fusion plasmids containing the individual promoters. The transcriptional activities of PI1 and PI2 did not change as the growth rate was varied from 0.3 to 1.7 doublings per h. In contrast, a fusion plasmid carrying the rrnB P1 promoter displayed the expected growth rate response. The steady-state concentrations of infC mRNA in cells grown at different rates were measured and found not to vary. These results indicate that the previously reported growth rate regulation of IF3 biosynthesis neither is accomplished by transcriptional control nor is a result of differential mRNA stability. In view of these results, the steady-state levels of IF3 in cells grown at a number of different growth rates were determined by quantitative immunoblotting. IF3 levels were found to vary with growth rate in a manner essentially identical to that observed for ribosomes. A model accounting for these results and describing a mechanism for coordinate growth rate-regulated expression of ribosomes and IF3 is presented.

Lyme disease is a tickborne zoonosis that occurs globally in the Northern Hemisphere [1, 2]. In North America, Lyme disease appears to be caused exclusively by Borrelia burgdorferi sensu stricto, whereas in Europe other species of Borrelia predominate [3–6]. A variety of typing systems exist to distinguish genetically distinct strains of B. burgdorferi [3]. On the basis of restriction fragment–length polymorphism (RFLP) of the 16S–23S rRNA intergenic spacer, B. burgdorferi has been classified into 3 distinct genetic subgroups (herein called genotypes), arbitrarily called ribosomal spacer type (RST) 1, RST2, and RST3 [7, 8]. Greater separation into distinct genotypes is achievable on the basis of the sequence heterogeneity of the outer surface protein C gene (ospC) [9, 10]. Genetically distinct genotypes of B. burgdorferi appear to differ ecologically and epidemiologically [11–14], suggesting that genotype classification is relevant to understanding the basic biology of the spirochete. Differential pathogenicity of B. burgdorferi on the basis of genotype has been reported in several studies. For example, in an analysis of 104 borrelial strains recovered from the skin of patients with a diagnosis of erythema migrans in Westchester County, New York, it was found that patients with disseminated infection (i.e., positive blood culture and/or multiple erythema migrans skin lesions) were at least 5 times more likely to have been infected with RST1 strains of B. burgdorferi than with RST3 strains [15]. A subsequent study of patients from Connecticut and Rhode Island with erythema migrans similarly found that infections believed to have disseminated were 5 times more common among RST1-infected patients compared with those infected with RST3 strains of B. burgdorferi [16]. In a study from New York State, dissemination of B. burgdorferi to blood or cerebrospinal fluid (CSF) was exclusively associated with ospC genotypes A, B, I, or K [9]. Subsequent studies from other geographic areas, however, have suggested that hematogenous dissemination is not restricted to just these 4 ospC genotypes [16–18]. The present study was undertaken to evaluate the relationship between RST and ospC genotypes and to examine the association between specific genotypes and objective evidence of dissemination of the spirochete in patients, using the largest sample of clinical isolates of B. burgdorferi studied to date. In addition, the frequency distribution of RST and ospC genotypes in the skin or blood of patients with culture-confirmed erythema migrans was compared with that present in local tick populations.

Lyme borreliosis, the most commonly reported vector-borne disease in North America, is caused by the spirochete Borrelia burgdorferi . Given the extensive genetic polymorphism of B . burgdorferi , elucidation of the population genetic structure of the bacterium in clinical samples may be relevant for understanding disease pathogenesis and may have applicability for the development of diagnostic tests and vaccine preparations. In this investigation, the genetic polymorphism of the 16S-23S rRNA ( rrs - rrlA ) intergenic spacer and ospC was investigated at the sequence level in 127 clinical isolates obtained from patients with early Lyme borreliosis evaluated in suburban New York City. Sixteen distinct rrs - rrlA and 16 distinct ospC alleles were identified, representing virtually all of the genotypes previously found in questing Ixodes scapularis nymphs in this region. In addition, a new ospC group was identified in a single patient. The strong linkage observed between the chromosome-located rrs - rrlA and plasmid-borne ospC genes suggests a clonal structure of B . burgdorferi in these isolates, despite evidence of recombination at ospC .

Lyme disease, which is caused by the spirochete Borrelia burgdorferi, is the most common vectorborne disease in North America [1, 2]. Patients with B. burgdorferi infection may have no symptoms or may present with a wide range of clinical manifestations involving the skin, joints, nervous system, or heart [3]. There is increasing evidence that genotypic variation of B. burgdorferi accounts for the variability in the microorganism’s pathogenicity [4–9]. On the basis of a polymerase chain reaction–restriction fragment–length polymorphism analysis (PCR-RFLP) of the 16S–23S ribosomal DNA intergenic spacer, clinical isolates of B. burgdorferi from the United States can be classified genetically into 3 distinct genotypes designated as RST1, RST2, and RST3 [10, 11]. In clinical studies of patients with Lyme disease–associated erythema migrans, RST1 was the most pathogenic, and RST3 was the least pathogenic. Compared with infection with RST2 or RST3 strains, infection with RST1 strains has been associated with a greater number of spirochetes in the erythema migrans skin lesion [6] and a higher rate of both spirochetemia and development of secondary skin lesions [4]. Whether human host factors contribute to the risk of infection or affect the clinical presentation of infection with strains from particular genotypes of B. burgdorferi is unknown. To explore this possibility, we studied the HLA class II alleles in patients with Lyme disease–associated erythema migrans and in relation to the genotype of the infecting B. burgdorferi strain. Our hypothesis was that HLA class II alleles might either influence susceptibility to infection with strains from the various genotypes of B. burgdorferi or affect the clinical manifestations. More specifically, we tested the possibility that either (1) genotype RST1 strains appear to be more invasive because they more commonly infect patients whose carriage of certain HLA class II alleles makes them less able to control RST1 infection or (2) infection with the least invasive RST3 strains is restricted to patients with certain HLA class II alleles.

Functions of the Borrelia burgdorferi RecA protein were investigated in Escherichia coli recA null mutants. Complementation with B. burgdorferi recA increased survival of E. coli recA mutants by 3 orders of magnitude at a UV dose of 2,000 μJ/cm 2 . The viability at this UV dose was about 10% that provided by the homologous recA gene. Expression of B. burgdorferi recA resulted in survival of E. coli at levels of mitomycin C that were lethal to noncomplemented hosts. B. burgdorferi RecA was as effective as E. coli RecA in mediating homologous recombination in E. coli . Furthermore, E. coli λ phage lysogens complemented with B. burgdorferi recA produced phage even in the absence of UV irradiation. The level of phage induction was 55-fold higher than the level in cells complemented with the homologous recA gene, suggesting that B. burgdorferi RecA may possess an enhanced coprotease activity. This study indicates that B. burgdorferi RecA mediates the same functions in E. coli as the homologous E. coli protein mediates. However, the rapid loss of viability and the absence of induction in recA expression after UV irradiation in B. burgdorferi suggest that recA is not involved in the repair of UV-induced damage in B. burgdorferi . The primary role of RecA in B. burgdorferi is likely to be a role in some aspect of recombination.

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi -specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B . burgdorferi , in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B . burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I . scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states ( P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B . burgdorferi ( P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B . burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.

Variability of disease manifestations has been noted in patients with Lyme disease. A contributing factor to this variation may be the number of spirochetes present in infected patients. We evaluated clinical and laboratory findings for patients with erythema migrans with regard to the number of Borrelia burgdorferi organisms detected by quantitative PCR (qPCR) in 2-mm skin biopsy specimens. B. burgdorferi was detected in 80% (40 of 50) of the specimens tested; the mean number of spirochetes in these specimens ranged over 3 orders of magnitude (10 to 11,000 spirochetes per 2-mm biopsy specimen). Larger numbers of spirochetes were significantly associated with a shorter duration of the erythema migrans skin lesion ( P = 0.020), smaller skin lesions ( P = 0.020), and infection with a specific genotype of B. burgdorferi ( P = 0.008) but not with the number or severity of symptoms. Skin culture positivity was significantly associated with skin lesions containing larger numbers of spirochetes ( P = 0.019).

The pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing 2 distinct ribosomal DNA spacer restriction fragment–length polymorphism genotypes (RSTs) was assessed in a murine model of Lyme disease. B. burgdorferi was recovered from 71.5% and 26.6% of specimens from mice infected with RST1 and RST3 isolates, respectively (P < .0001). The average ankle diameter and histologic scores for carditis and arthritis were significantly higher after 2 weeks of infection among mice infected with RST1 isolates than among those infected with RST3 isolates (P < .001). These clinical manifestations were associated with larger numbers of spirochetes in target tissues but not with the serum sensitivity of the individual isolates. Thus, the development and severity of disease in genetically identical susceptible hosts is determined mainly by the pathogenic properties of the infecting B. burgdorferi isolate. The RST1 genotype is genetically homogeneous and thus may represent a recently evolved clonal lineage that is highly pathogenic in humans and animals.

Erythematous rashes that are suggestive of early Lyme disease have been associated with the bite of Amblyomma americanum ticks, particularly in the southern United States. However, Borrelia burgdorferi, the causative agent of Lyme disease, has not been cultured from skin biopsy specimens from these patients, and diagnostic serum antibodies usually have not been found. Borrelia lonestari sp nov, an uncultured spirochete, has been detected in A. americanum ticks by DNA amplification techniques, but its role in human illness is unknown. We observed erythema migrans in a patient with an attached A. americanum tick. DNA amplification of the flagellin gene flaB produced B. lonestari sequences from the skin of the patient that were identical to those found in the attached tick. B. lonestari is a probable cause of erythema migrans in humans.

Two hundred seventeen isolates of Borrelia burgdorferi originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of B. burgdorferi performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [ P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.

To the Editor: Human granulocytic ehrlichiosis (HGE), first described in 1994 (1), is the second-most common tick-borne disease in the United States; Lyme disease is the most prevalent. Both diseases are transmitted by blacklegged ticks (Ixodes scapularis Say) that are abundant in southern New York state (2). Factors that influence the risk for infection, particularly the role of wildlife in transmitting the etiologic agent, Anaplasma (formerly Ehrlichia) phagocytophilum (3,4), to vector ticks are not well understood. In the absence of transovarial transmission (5,6), acquisition of A. phagocytophilum by its vector must result either from feeding on the blood of reservoir-competent hosts or by cofeeding of uninfected ticks in close proximity to infected ticks (7).

Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.