title: Chitobiose utilization in Borrelia burgdorferi is dually regulated by RpoD and RpoS.
abstract: BACKGROUND
  Borrelia burgdorferi has limited biosynthetic capabilities and must scavenge N-acetylglucosamine (GlcNAc), an essential component of the microbial cell wall, from the surrounding environment. Spirochetes cultured in the absence of free GlcNAc exhibit biphasic growth; however, addition of chitobiose (a dimer of GlcNAc) substitutes for free GlcNAc resulting in a single exponential phase. We evaluated the effect of RpoS and RpoN, the only alternative sigma factors in B. burgdorferi, on biphasic growth and chitobiose utilization in the absence of free GlcNAc. In addition, we investigated the source of GlcNAc in the second exponential phase.
  
  
  RESULTS
  By comparing the growth of wild-type cells to insertional mutants for rpoS and rpoN we determined that RpoS, but not RpoN, partially regulates both biphasic growth and chitobiose utilization. The rpoS mutant, cultured in the absence of free GlcNAc, exhibited a significant delay in the ability to initiate a second exponential phase compared to the wild type and rpoS complemented mutant. Expression analysis of chbC, which encodes the membrane-spanning protein of the chitobiose phosphotransferase system, suggests the delay is due to the inability of the rpoS mutant to up regulate chbC. Furthermore, supplementing GlcNAc starved cultures with high concentrations (75 or 150 microM) of chitobiose resulted in biphasic growth of the rpoS mutant compared to a single exponential phase for the wild type and rpoS complemented mutant. In contrast, growth of the rpoN mutant under all conditions was similar to the wild type. 5' Rapid amplification of cDNA ends (5' RACE) revealed the transcriptional start site for chbC to be 42 bp upstream of the translational start site. Analysis of the chbC promoter region revealed homology to previously described RpoD and RpoS B. burgdorferi promoters. We also determined that yeastolate, a component of the growth medium (BSK-II), is not essential for second exponential phase growth.
  
  
  CONCLUSIONS
  Together these results suggest that RpoD and RpoS, but not RpoN, regulate biphasic growth and chitobiose utilization in B. burgdorferi by regulating the expression of the chitobiose transporter (chbC). The data also demonstrate that the second exponential phase observed in wild-type cells in the absence of free GlcNAc is not due to free chitobiose or GlcNAc oligomers present in the medium.
authors.affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA. rrhodes@mail.uri.edu
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Rhodes
authors.forename: Wendy
authors.initials: W
authors.lastname: Coy
authors.forename: David R
authors.initials: DR
authors.lastname: Nelson
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D025065:Borrelia burgdorferi / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D003470:Culture Media; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004187:Disaccharides / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site
chemical_list: D001426:Bacterial Proteins; C436306:ChbC protein, Borrelia burgdorferi; D003470:Culture Media; D004187:Disaccharides; D026901:Membrane Transport Proteins; D012329:RNA, Bacterial; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; C032438:chitobiose; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D000117:Acetylglucosamine