Characterization of the RelBbu 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'-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. 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°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°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 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.

Chemical sensing in mammalian host-bacterial commensal associations.

The mammalian gastrointestinal (GI) tract is colonized by a complex consortium of bacterial species. Bacteria engage in chemical signaling to coordinate population-wide behavior. However, it is unclear if chemical sensing plays a role in establishing mammalian host-bacterial commensal relationships. Enterohemorrhagic Escherichia coli (EHEC) is a deadly human pathogen but is a member of the GI flora in cattle, its main reservoir. EHEC harbors SdiA, a regulator that senses acyl-homoserine lactones (AHLs) produced by other bacteria. Here, we show that SdiA is necessary for EHEC colonization of cattle and that AHLs are prominent within the bovine rumen but absent in other areas of the GI tract. We also assessed the rumen metagenome of heifers, and we show that it is dominated by Clostridia and/or Bacilli but also harbors Bacteroidetes. Of note, some members of the Bacteroidetes phyla have been previously reported to produce AHLs. SdiA-AHL chemical signaling aids EHEC in gauging these GI environments, and promotes adaptation to a commensal lifestyle. We show that chemical sensing in the mammalian GI tract determines the niche specificity for colonization by a commensal bacterium of its natural animal reservoir. Chemical sensing may be a general mechanism used by commensal bacteria to sense and adapt to their mammalian hosts. Additionally, because EHEC is largely prevalent in cattle herds, interference with SdiA-mediated cattle colonization is an exciting alternative to diminish contamination of meat products and cross-contamination of produce crops because of cattle shedding of this human pathogen.

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')-IIIa from Streptococcus faecalis, aad9 from Staphylococcus aureus Tn554, linA' 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')-IIIa, aad9, linA' 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.