Characterization of the Rel(Bbu) Regulon in Borrelia burgdorferi Reveals Modulation of Glycerol Metabolism by (p)ppGpp

The bacterial stringent response is triggered by deficiencies of available nutrients and other environmental stresses. It is mediated by 5\u27-triphosphate-guanosine-3\u27-diphosphate and 5\u27-diphosphate-guanosine-3\u27-diphosphate (collectively (p)ppGpp) and generates global changes in gene expression and metabolism that enable bacteria to adapt to and survive these challenges. Borrelia burgdorferi encounters multiple stressors in its cycling between ticks and mammals that could trigger the stringent response. We have previously shown that the B. burgdorferi stringent response is mediated by a single enzyme, RelBbu, with both (p)ppGpp synthase and hydrolase activities, and that a B. burgdorferi 297 relBbu null deletion mutant was defective in adapting to stationary phase, incapable of down-regulating synthesis of rRNA and could not infect mice. We have now used this deletion mutant and microarray analysis to identify genes comprising the rel regulon in B. burgdorferi cultured at 34 degrees C, and found that transcription of genes involved in glycerol metabolism is induced by relBbu. Culture of the wild type parental strain, the relBbu deletion mutant and its complemented derivative at 34 degrees C and 25 degrees C in media containing glucose or glycerol as principal carbon sources revealed a growth defect in the mutant, most evident at the lower temperature. Transcriptional analysis of the glp operon for glycerol uptake and metabolism in these three strains confirmed that relBbu was necessary and sufficient to increase transcription of this operon in the presence of glycerol at both temperatures. These results confirm and extend previous findings regarding the stringent response in B. burgdorferi. They also demonstrate that the stringent response regulates glycerol metabolism in this organism and is likely crucial for its optimal growth in ticks

GABA-modulating bacteria of the human gut microbiota.

The gut microbiota affects many important host functions, including the immune response and the nervous system1. However, while substantial progress has been made in growing diverse microorganisms of the microbiota2, 23-65% of species residing in the human gut remain uncultured3,4, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence in artificial media of key growth factors that are provided by neighbouring bacteria in situ5,6. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bioassay-driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter GABA (γ-aminobutyric acid). GABA was the only tested nutrient that supported the growth of KLE1738, and a genome analysis supported a GABA-dependent metabolism mechanism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome-based metabolic modelling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. A transcriptome analysis of human stool samples from healthy individuals showed that GABA-producing pathways are actively expressed by Bacteroides, Parabacteroides and Escherichia species. By coupling 16S ribosmal RNA sequencing with functional magentic resonance imaging in patients with major depressive disorder, a disease associated with an altered GABA-mediated response, we found that the relative abundance levels of faecal Bacteroides are negatively correlated with brain signatures associated with depression

Comparative Genome Hybridization Reveals Substantial Variation among Clinical Isolates of Borrelia burgdorferi Sensu Stricto with Different Pathogenic Properties

Clinical and murine studies suggest that there is a differential pathogenicity of different genotypes of Borrelia burgdorferi, the spirochetal agent of Lyme disease. Comparative genome hybridization was used to explore the relationship between different genotypes. The chromosomes of all studied isolates were highly conserved (>93%) with respect to both sequence and gene order. Plasmid sequences were substantially more diverse. Plasmids lp54, cp26, and cp32 were present in all tested isolates, and their sequences and gene order were conserved. The majority of linear plasmids showed variation both in terms of presence among different isolates and in terms of sequence and gene order. The data strongly imply that all B. burgdorferi clinical isolates contain linear plasmids related to each other, but the structure of these replicons may vary substantially from isolate to isolate. These alterations include deletions and presumed rearrangements that are likely to result in unique plasmid elements in many isolates. There is a strong correlation between complete genome hybridization profiles and other typing methods, which, in turn, also correlate to differences in pathogenicity. Because there is substantially less variation in the chromosomal and circular plasmid portions of the genome, the major differences in open reading frame content and genomic diversity among isolates are linear plasmid driven

Erythromycin Resistance in Borrelia burgdorferi

Susceptibility testing of laboratory strains and clinical isolates of Borrelia burgdorferi indicates that resistance to erythromycin is present in them. Evaluation of the MICs, minimal bactericidal concentrations, and kinetics of bacterial killing of erythromycin suggests that this resistance is increased by preexposure to the antibiotic, is dependent on inoculum size, and may be the result of selection of subpopulations of bacterial cells with increased resistance

BB0844, an RpoS-Regulated Protein, Is Dispensable for Borrelia burgdorferi Infectivity and Maintenance in the Mouse-Tick Infectious Cycle▿

The genome of Borrelia burgdorferi, the causative agent of Lyme disease, is comprised of a large linear chromosome and numerous smaller linear and circular plasmids. B. burgdorferi exhibits substantial genomic variation, and previous studies revealed genotype-specific variation at the right chromosomal telomere. A correlation has also been established between genotype and invasiveness. The correlation between chromosome length and genotype and between genotype and invasiveness suggested that a gene(s) at the right chromosome telomere may be required for virulence. Of particular interest was bb0844, an RpoS-regulated gene at the right telomere, the expression of which is induced when the spirochete undergoes adaptation to the mammalian host. The structure of the right chromosomal telomere was examined in 53 B. burgdorferi clinical isolates of various genotypes. Four distinct patterns were observed for bb0844: (i) chromosomal localization, (ii) plasmid localization, (iii) presence on both chromosome and plasmid, and (iv) complete absence. These patterns correlated with the B. burgdorferi genotype. On the basis of available sequence data, we propose a mechanism for the genomic rearrangements that accounts for the variability in bb0844 genomic localization. To further explore the role of BB0844 in the spirochete life cycle, a bb0844 deletion mutant was constructed by allelic exchange, and the viability of wild-type and bb0844 deletion mutants was examined in an experimental mouse-tick infection model. The bb0844 mutant was fully infectious in C3H/HeJ mice by either needle inoculation or tick transmission with B. burgdorferi-infected Ixodes scapularis larvae. Naïve larval ticks acquired both wild-type and mutant spirochetes with equal efficiency from B. burgdorferi-infected mice. The results demonstrate that BB0844 is not required for spirochete viability, pathogenicity, or maintenance in the tick vector or the mammalian host. At present, a defined role for BB0844 in B. burgdorferi cannot be ascertained

Anxiety and type 1 diabetes management: guardian and child report in a pediatric endocrinology clinic

Background: Childhood anxiety prevents optimal diabetes management yet may be underrecognized by guardians. Objective: We aimed to investigate associations among anxiety, diabetes treatment adherence, and diabetes symptom control through child and guardian report. Methods: Cross-sectional pilot study surveying a convenience sample of children (ages 2–21) in a pediatric endocrinology clinic. Behavior Assessment System for Children, Second Edition 2, Self-Care Inventory Report, and Pediatric Quality of Life measured anxiety, diabetes treatment adherence, and diabetes symptom control. Analyses were performed with Spearman correlations. Results: Prevalence of anxiety and related behaviors was higher when reported by children (13% and 24%) vs. guardians (5% and 13%). Child-reported anxiety was associated with worse symptom control in all ages (Pediatric Quality of Life [r = −0.55, P 12 (rho = 0.686, P = 0.003)—although not significantly for children ≤ 12 (rho = 0.201, P = 0.473). Conclusion: Anxiety in children with type 1 diabetes varies with the domain of diabetes management (treatment adherence vs. symptom control) and reporting source (child vs. guardian). Children aged ≤12 exhibited a stronger relationship between higher anxiety and worse diabetes management with worse treatment adherence and symptom control in the presence of higher anxiety. Guardians of younger children were less effective at recognizing symptoms. Challenges identifying anxiety and its detrimental effects on diabetes management suggest routine screening of anxiety in pediatric endocrinology clinics is especially salient

Novel Antibiotic-Resistance Markers in pGK12-Derived Vectors for Borrelia Burgdorferi

Extension of molecular genetics studies in Borrelia burgdorferi has been hampered by a lack of a variety of antibiotic resistance selective markers. Such markers are critical for isolation of B. burgdorferi strains with multiple mutants, for complementation with different cloning vectors, and for methods such as negative selection and reporter genes. To remedy this lack, resistance to various antibiotics of non-infectious (B31, 297) and infectious (N40) B. burgdorferi strains was examined and vectors incorporating appropriate antibiotic resistance genes as selective markers were developed. Minimal inhibitory concentrations for growth of B. burgdorferi on plates and in liquid media for aminoglycosides (kanamycin, gentamycin, sisomycin, amikacin, spectinomycin, neomycin), macrolides-lincosamids (erythromycin, lincomycin), coumarin derivatives (coumermycin A(1), novobiocin), glycopeptides (vancomycin, ristocetin), peptides (bacitracin, cycloserine), and chloramphenicol were found to differ significantly. There were also striking differences in resistance to these antibiotics between non-infectious and infectious B. burgdorferi strains. Antibiotic-resistance genes aph(3\u27)-IIIa from Streptococcus faecalis, aad9 from Staphylococcus aureus Tn554, linA\u27 from Staphylococcus aureus, and aac(3)-VIa from Enterobacter cloacae (conferring resistance to kanamycin, spectinomycin, lincomycin, and gentamycin/sisomycin, respectively) were subcloned either with their own promoters or under the control of the B. burgdorferi flaB promoter into pGK12 or its derivative pED1 to develop new cloning vectors for B. burgdorferi with the rationale that the absence of homologous regions between derived recombinant plasmids lacking the flaB promoter and the B. burgdorferi genome would permit avoidance of possible recombination with target DNA. Resistance to the corresponding antibiotic was conferred by vectors containing aph(3\u27)-IIIa, aad9, linA\u27 or aac(3)-VIa whether under the control of their own promoters or under the control of the flaB promoter. We conclude that these markers can be used for genetic study of B. burgdorferi and suggest they will be an important addition to the previously used coumermycin A(1), erythromycin and kanamycin in these studies

Characterization of the Rel_Bbu Regulon in <i>Borrelia burgdorferi</i> Reveals Modulation of Glycerol Metabolism by (p)ppGpp

<div><p>The bacterial stringent response is triggered by deficiencies of available nutrients and other environmental stresses. It is mediated by 5'-triphosphate-guanosine-3'-diphosphate and 5'-diphosphate-guanosine-3'-diphosphate (collectively (p)ppGpp) and generates global changes in gene expression and metabolism that enable bacteria to adapt to and survive these challenges. <i>Borrelia burgdorferi</i> encounters multiple stressors in its cycling between ticks and mammals that could trigger the stringent response. We have previously shown that the <i>B. burgdorferi</i> stringent response is mediated by a single enzyme, Rel_Bbu, with both (p)ppGpp synthase and hydrolase activities, and that a <i>B. burgdorferi</i> 297 rel_Bbu null deletion mutant was defective in adapting to stationary phase, incapable of down-regulating synthesis of rRNA and could not infect mice. We have now used this deletion mutant and microarray analysis to identify genes comprising the <i>rel</i> regulon in <i>B. burgdorferi</i> cultured at 34°C, and found that transcription of genes involved in glycerol metabolism is induced by <i>rel</i>_Bbu. Culture of the wild type parental strain, the <i>rel</i>_Bbu deletion mutant and its complemented derivative at 34°C and 25°C in media containing glucose or glycerol as principal carbon sources revealed a growth defect in the mutant, most evident at the lower temperature. Transcriptional analysis of the <i>glp</i> operon for glycerol uptake and metabolism in these three strains confirmed that <i>rel</i>_Bbu was necessary and sufficient to increase transcription of this operon in the presence of glycerol at both temperatures. These results confirm and extend previous findings regarding the stringent response in <i>B. burgdorferi</i>. They also demonstrate that the stringent response regulates glycerol metabolism in this organism and is likely crucial for its optimal growth in ticks.</p></div

Transcriptional analysis by qRT-PCR of selected genes in <i>B</i>. <i>burgdorferi</i> 297 Δ<i>rel</i>_<i>Bbu</i> relative to expression of these genes in the wild type parental strain during (A) exponential and (B) stationary phases of growth in BSK-H at 34°C.

<p>*, P < 0.02; **, P < 0.005; ***, P < 0.001. Genes selected either had an assigned function and were regulated in operons, were involved in various aspects of cellular metabolism, were regulatory in nature or were localized to the cell envelope. Increased expression of genes in the mutant relative to wild type is consistent with <i>rel</i>-mediated repression of these genes in the wild type, while decreased expression is consistent with <i>rel</i>-mediated induction in the wild type. RT-PCR data were discordant with microarrays for <i>potA</i>, <i>mvaA</i>, <i>rpoD</i>, <i>apt</i>, BBB07, and <i>blyA</i> in the exponential phase of growth, and for <i>oppF</i> in the stationary phase of growth. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118063#sec009" target="_blank">Materials and Methods</a> for details of transcriptional analyses.</p

Transcriptional analysis of <i>glp</i> operon genes in wild type <i>B</i>. <i>burgdorferi</i> 297 (solid bars), Δ<i>rel</i>_<i>Bbu</i> (open bars), and Δ<i>rel</i>_<i>Bbu</i> complemented with pKFSS1-Δ<i>rel</i>_<i>Bbu</i> (grey bars) during late logarithmic growth phase in BSK-Lite medium containing glucose or glycerol as principal carbon sources.

<p>Glucose-containing medium, 34°C (A). Glycerol-containing medium, 34°C (B). Glucose-containing medium, 25°C (C). Glycerol-containing medium, 25°C (D).</p