title: CrdR function in a curdlan-producing Agrobacterium sp. ATCC31749 strain.
abstract: BACKGROUND
  Agrobacterium sp. ATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation. The helix-turn-helix transcriptional regulatory protein (crdR) essential for curdlan production has been analyzed, but whether crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC) is uncertain. To elucidate the molecular function of crdR in curdlan biosynthesis, we constructed a crdR knockout mutant along with pBQcrdR and pBQNcrdR vectors with crdR expression driven by a T5 promoter and crdR native promoter, respectively. Also, we constructed a pAG with the green fluorescent protein (GFP) gene driven by a curdlan biosynthetic operon promoter (crdP) to measure the effects of crdR expression on curdlan biosynthesis.
  
  
  RESULTS
  Compared with wild-type (WT) strain biomass production, the biomass of the crdR knockout mutant was not significantly different in either exponential or stationary phases of growth. Mutant cells were non-capsulated and planktonic and produced significantly less curdlan. WT cells were curdlan-capsulated and aggregated in the stationery phase. pBQcrdR transformed to the WT strain had a 38% greater curdlan yield and pBQcrdR and pBQNcrdR transformed to the crdR mutant strain recovered 18% and 105% curdlan titers of the WT ATCC31749 strain, respectively. Consistent with its function of promoting curdlan biosynthesis, curdlan biosynthetic operon promoter (crdP) controlled GFP expression caused the transgenic strain to have higher GFP relative fluorescence in the WT strain, and no color change was observed with low GFP relative fluorescence in the crdR mutant strain as evidenced by fluorescent microscopy and spectrometric assay. q-RT-PCR revealed that crdR expression in the stationary phase was greater than in the exponential phase, and crdR overexpression in the WT strain increased crdA, crdS, and crdC expression. We also confirmed that purified crdR protein can specifically bind to the crd operon promoter region, and we inferred that crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC).
  
  
  CONCLUSIONS
  CrdR is a positive transcriptional regulator of the crd operon for promoting curdlan biosynthesis in ATCC31749. The potential binding region of crdR is located within the -98 bp fragment upstream from the crdA start codon.
authors.affiliation: College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, China. 14787884558@163.com.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Yu
authors.affiliation: College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, China. zc876827281@sina.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.affiliation: College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, China. yangmumuxi@sina.cn.
authors.forename: Liping
authors.initials: L
authors.lastname: Yang
authors.affiliation: College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, China. 908039065@qq.com.
authors.forename: Lamei
authors.initials: L
authors.lastname: Zhao
authors.affiliation: College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, China. linchun0303@163.com.
authors.forename: Chun
authors.initials: C
authors.lastname: Lin
authors.affiliation: College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, China. lzj1022@163.com.
authors.forename: Zhengjie
authors.initials: Z
authors.lastname: Liu
authors.affiliation: College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, China. mao2010zichao@126.com.|National and Local Joint Engineering Research Center for Screening and Application of Microbial Strains, Kunming, China. mao2010zichao@126.com.
authors.forename: Zichao
authors.initials: Z
authors.lastname: Mao
mesh_terms: D060054:Agrobacterium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003062:Codon / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009584:Nitrogen / Q000172:deficiency; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic; D047071:beta-Glucans / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D047071:beta-Glucans; D049452:Green Fluorescent Proteins; C038459:curdlan; D009584:Nitrogen