Abstract
BackgroundAgrobacterium 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.ResultsCompared 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).ConclusionsCrdR 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 codonElectronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0356-1) contains supplementary material, which is available to authorized users.
Highlights
IntroductionATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation
Compared with wild-type (WT) ATCC31749, which is capsulated in the stationary phase, the crdR knockout strain (ATCC31749ΔcrdR) produced less curdlan (Figures 3 and 4) leading to motile and non-capsulated planktonic forms (Figure 3) in both exponential and stationary phases. crdR expression driven by promoters of T5 and native crdR in both ATCC31749 and ATCC31749ΔcrdR strains, respectively, were obtained by transforming the constructs of pBQcrdR and pBQNcrdR (Figure 1)
Expression of curdlan biosynthesis genes responding to crdR overexpression Because crdR is an important regulator of curdlan biosynthesis (Figures 3 and 4), we investigated whether crdR activates expression of crd operon genes. q-RTPCR analysis was used to evaluate the effects of crdR on crdA, crdS, and crdC mRNA
Summary
ATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation. 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. A water insoluble β-D-1, 3-glucan, can be efficiently produced by Agrobacterium sp. ATCC31749 during stressors of low pH and nitrogen starvation [2,3,4]. Many cultivating conditions including low pH [18], limited nitrogen [19], high dissolved oxygen [20] and adding uracil or cytosine and phosphate salts [21,22,23] influence curdlan biosynthesis and accumulation.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
