CrdR function in a curdlan-producing Agrobacterium sp. ATCC31749 strain
© Yu et al.; licensee BioMed Central. 2015
Received: 6 October 2014
Accepted: 20 January 2015
Published: 10 February 2015
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.
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).
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
KeywordscrdR Curdlan Agrobacterium Transcriptional regulator
Microbes can produce diverse extracellular polysaccharides (EPS) for survival in harsh conditions . Curdlan, a water insoluble β-D-1, 3-glucan, can be efficiently produced by Agrobacterium sp. ATCC31749 during stressors of low pH and nitrogen starvation [2-4]. Because of its special gel and immunomodulatory properties, curdlan and its derivatives can be used as food additives and in pharmaceutic products [5-7]. β-D-1,3-glucans can be synthesized by bacteria, fungi  and plants ; however, large-scale curdlan production occurs mainly via fermentation in Agrobacterium [3,10], Rhizobium strains  and Cellulomonas flavigena . An efficient curdlan-producing strain, ATCC31749, whose draft genome sequence is more than 95% homologous to the Agrobacterium tumefaciens strain C58 (ATCC33970) genome, is regarded as a model organism for elucidating curdlan biosynthetic pathways and regulatory mechanisms [13,14]. Using chemical mutant selection, the curdlan biosynthesis operon (crd) was found to contain crdA, crdS, and crdC genes in the ATCC31749 strain [15-17]. Many cultivating conditions including low pH , limited nitrogen , high dissolved oxygen  and adding uracil or cytosine and phosphate salts [21-23] influence curdlan biosynthesis and accumulation. However, how curdlan biosynthesis gene expression is regulated is unclear.
ATCC31750, a mutant strain derived from ATCC31749, had significantly altered intracellular proteins with changes in pH. Specifically, at pH 5.5 (compared to 7.0), key enzymes of curdlan biosynthesis, such as the catalytic subunit of β-1,3-glucan synthase (crdS), UTP-glucose-1-phosphate uridylytransferase (galU), and phosphoglucomutase (pgm) were increased 10, 3, and 17 times, respectively . Intracellular pH changes may activate synthesis of a cellular stringent response signal (p)ppGpp to alter formation of acidocalcisome, which helps maintain intracellular pH and ion homeostasis . To learn how low pH affects curdlan biosynthesis in an ATCC31749 strain, we analyzed genomic sequences of ATCC31749 (access No: AECL01000001–AECL01000095) and that of Sinorhizobium meliloti (access No: NC_003047), which is an acid-tolerant, symbiotic nitrogen-fixing strain  using BLAST alignment. We found a transcriptional regulator, PhrR (access No: NC_003047.1 (445435–445854), expression of this gene increased 5–6 times under conditions of low pH (pH 6.2) in S. meliloti . The PhrR gene has a homologous counterpart, AGRO_0435, in ATCC31749. Both PhrR and AGRO_0435 are helix-turn-helix transcriptional factors of the XRE-family, which includes HipB of Escherichia coli (E coli), CH00371 of Rhizobium Leguminosarum (R. Leguminosarum), and PraR of Azorhizobium Caulinodans (A. caulinodans) (Additional file 1) [27-35]. The existence of an essential curdlan production regulatory locus other than the crd operon—locus II—was suggested by Stasinopoulos’s group  DNA sequencing confirmed that the locus II gene encodes a helix-turn-helix transcriptional regulatory protein, crdR, and that AGRO_0435 is the crdR gene , Unfortunately, whether crdR acts directly to regulate crdASC expression is unclear, so we investigated the role of crdR on crdASC transcriptional activation.
Bacterial strains and vectors used
Bacterial strains and plasmids used in this study
Res − Mod − ompT (DE3 with T7 pol) (pLysS with T7 lysozyme;Cm r) Novagen
Curdlan-producing Agrobacterium sp. (wild-type strain)
ATCC 31749 mutant with gene knockout of crdR
GFP expression vector
Expression vector, Amp R
Gram-negative broad host vector
Expression vector carrying sacBR, Gm R
Vector derived from both pQE80 and pBBR122
Suicide vector for crdR knock-out
Expression vector with T 5 driving crdR expression
Expression vector with crdRP driving crdR expression
Expression vector with GFP driven by crd promoter
Derivative of pMD18-T with part of crdA
Derivative of pMD18-T with part of crdS
Derivative of pMD18-T with part of crdC
Derivative of pMD18-T with part of crdR
Primers used in this study
Knockout of the CrdR (AGRO_0435 gene)
Construction of pBQcrdR and pBQNcrdR for homogenous AGRO_0435 expression
A 437-bp full-length coding region of crdR was amplified with PCR using the primer pairs crdR-1 and crdR-2 (Table 2) with genomic ATCC31749 DNA. The amplified crdR fragment, digested with BamHI and SacI, was ligated into the pBQ vector , creating the pBQcrdR vector (Table 1). To construct the vector for crdR expression driven by its native promoter of crdR, an AGRO_0435 fragment with up- and down-stream flanking sequences was PCR cloned using primers NcrdR-1 and NcrdR-2 (Table 2) The obtained 1,302 bp PCR fragment which was double digested with both SacI and XhoI was inserted into SalI and sacI sites of pBQ to create pBQNcrdR (Table 1, Figure 1).
Construction of pAG vector with GFP expression driven by the crd operon promoter
The predicted crd promoter (crdP), which is a 607-bp fragment upstream from the start codon of crdA (ATG), was amplified from genomic ATCC31749 DNA with primers crdAPG-1 and crdAPG-2 (Table 2). The GFP code sequence was amplified with primers GFP-1 and GFP-2 (Table 2) from plasmid pEGFP (Clontech, Mountain View, CA) and the two fragments were fused via PCR amplification. The resultant fused fragment, digested with XhoI and BamHI, was inserted into the same sites of plasmid pQE81L to yield pQEAG. After digestion with SalI, the fragment containing the gram-negative broad host replicating origin and the kanamycin (Kan) resistant gene amplified from pBBR122 (Table 1) with primers pairs Rep-Kan-1 and Rep-Kan-2 (Table 2), was inserted into the XhoI site of pQEAG to yield pAG (Table 1).
Curdlan fermentation and yield analysis
A two-step fermentation protocol was used to measure curdlan yields. In brief, ATCC 31749 and modified strains were inoculated into test tubes containing 5 mL LB and grown overnight at 30°C with 200 revolutions per minute (rpm). About 2 mL each of the seed cultures (SC) were transferred into 500-mL flasks containing 100 mL LB with or without IPTG (final concentration 0.5 mM) at 25°C, 200 rpm for 4 h. Cells were collected by centrifugation (1000 × g for 10 min, 4°C) and cell pellets were added to 125 mL curdlan-producing medium in a 500-mL flask which was shaken at 200 rpm. Every 24 h for 5 days reaction, 15-mL samples were taken from the culture mixture and samples were centrifuged at 8,000 × g for 5 min to collect pellets. Pellets containing both cells and curdlan were resuspended in 15 mL NaOH solution (1 mol/L) for 2 h. Cells pellets were separated by centrifugation at 8,000 × g for 5 min and resulting curdlan was precipitated by the addition of 2.0 mol/L HCl and the pH was adjusted to 6.5. Curdlan was recovered by centrifugation, washed, and dried to a constant weight in an oven (80°C).
crdR, crdA, crdS, and crdC expression analysis using q-RT-PCR
Total RNA was extracted with an EasyPure RNA Kit (TransGen Biotech, Beijing, China), according to the manufacturer’s protocol. The quality and quantity of the extracted RNA was measured using an Ultrospec 2100 spectrophotometer (Amersham Biosciences, Pittsburgh PA, USA) at 260 nm. cDNA synthesis was performed with a PrimerScript RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instruction by using a 6-bp random primers set. Selected fragments of crdR, crdA, crdS, and crdC, which were amplified with primers qcrdR-1 & qcrdR-2, qcrdA-1 & qcrdA-2, qcrdS-1 & qcrdS-2, and qcrdC-1 & qcrdC-2 (Table 2), were ligated into pMD18-T vectors respectively. Then, using those constructs as standard copies, q-RT-PCR quantification was performed using an Applied Biosystems 7500 fast realtime PCR system (Applied Biosystems, Grand Island, NY ) with SYBR Premix E*TaqII (TaKaRa). All samples were run in triplicate .
The constructed vector (pAG) was transformed into both wild-type ATCC31749 and a crdR mutant (ATCC31749ΔcrdR). Transformed bacterial cells were grown in LB for 12 h and curdlan-producing medium for 72 h at 30°C. GFP expression was observed under an optical microscope (Zeiss Observer Berlin, Germany), equipped with epi-fluorescence. Simultaneously, with excitation of 450–490 nm light, Green fluorescence of GFP was measured by a fluorospectrophotometer F97Pro (FProd, Shanghai, China) to collect the data of the emission spectrum and relative fluorescence of cells harvested from both bacterial cell -growing and curdlan-producing phases.
Expression and purification of 6 His-tagged crdR protein
6-His-tagged crdR was expressed in E. coli BL21 through pBQcrdR transformation. The resultant strain grew at 37°C in LB medium (OD600 nm = 0.5–0.6), and crdR protein expression was induced by adding IPTG (final concentration = 0.5 mM). The culture was shaken at 30°C for 4 h at 220 rpm. Cells were harvested by centrifugation were immediately extracted or frozen at −80°C until they were used. 6-His-tagged protein was purified by affinity chromatography using One-Step His-Tagged Protein Miniprep Pack (TIANDZ, Shanghai, China) according to the manufacturer’s instruction. Purified crdR dissolved in elution buffer was dialyzed with dialysis buffer (100 mM KAc, 1 mM CaCl2, 1 mM MgAc2, 1 mM EDTA, and 1 mM dithiothreitol, 10% glycerol) overnight in a semi-permeable membrane. Protein concentration was measured using an improved Coomassie assay with bovine serum albumin (BSA) as standard.
DNA binding analysis of CrdR by EMSA
DNA fragments containing various lengths of the crd promoter (crdP, crdP142, crdP108, crdP98, crdP53, crdP13, and crdP1) and ~450 bp upstream of the start codon (ATG) of crdR (relA(AGRO_1479) celA (AGRO_4469), and crdS (AGRO_1848) named relAP celAP and crdSP were obtained by PCR amplification with primers listed in Table 2 respectively, those fragments were purified with a DNA gel extraction kit (Sangon Biotech) respectively according to the manufacturer’s protocol. A electrophoretic mobility shift (EMSA) binding assay was performed as previously described with slight modifications . Briefly, 10 μL of 0.25–0.50 mg/mL purified His-tagged crdR in 4× EMSA buffer (15 mM HEPES, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 1 μg/mL poly dI-dC) was incubated with 10 μL of different purified target DNA fragments (0.5 μM) in ddH2O at room temperature for 30 min. DNA-protein complexes were loaded onto a 2% agarose gel and separated at 80 V for 1.5 h, and the gel was stained with SYBR Green I and visualized with a UV trans-illuminator (Upland, CA).
crdR knockout mutant construction and phenotypes
Expression of curdlan biosynthesis genes responding to crdR overexpression
The expressions of the crd genes quantified by q-RT-PCR*
ATCC31749/pBQ crdR( S)
0.2373 ± 0.0635
1.0944 ± 0.0956
244.2038 ± 18.3645
923.5690 ± 29.6543
0.4050 ± 0.1022
2.7739 ± 0.1185
86.7645 ± 4.2966
176.6229 ± 9.5607
0.4972 ± 0.9503
2.2787 ± 0.1263
4.5239 ± 0.3371
14.5744 ± 1.0027
14.9112 ± 1.0359
654.8556 ± 20.4256
29.2068 ± 1.8553
193.1226 ± 11.2094
GFP expression controlled by the crd operon promoter (crdP)
crdR binding with’ different crd operon promoter regions
Here, we report that the crdR, a homolog of PhrR of S. meliloti can activate curdlan synthetic gene expression in Agrobaterium sp. ATCC31749. To our knowledge, ours is the first report to depict molecular functions of the crdR gene. Our data indicate that curdlan yield in an over-expressing crdR strain increased 38% compared to the WT strain. Also, pBQNcrdR transformed to the crdR mutant strain recovered 105% curdlan synthesis of the WT strain (Figure 4). Also, when pAG was transformed into both crdR mutant and WT strains GFP expression controlled by the crd promoter was undetectable by fluorescent microscopy with low relative fluorescence in the crdR mutant. In contrast, the WT strain had visible green color with high relative fluorescence. Finally, q-RT-PCR analysis indicated that crdR is highly expressed in the stationary phase and that overexpression of crdR in the WT strain significantly increased expression of crdA, crdS and crdC. These data agree with previous reports that crdR is key for regulating curdlan biosynthesis. Purified crdR from E. coli BL21 can also specifically bind to the promoter region of crd offering initial evidence that crdR is a positive transcriptional regulator of the crd operon in ATCC31749.
The biomass accumulation in crdR mutant strains was not significantly different from the WT strain, suggesting that the crdR gene is not required for cell growth. Microscopic observation revealed that the crdR mutant was nearly curdlan deficient, resulting in mutant cells with non-capsulated planktonic forms. The WT ATCC31749 strain and the complementary strain of the crdR mutant, ATCC31749ΔcrdR/pBQNcrdR, accumulated curdlan in the stationary phase in culture media with low pH and limited nitrogen, leading to cells were capsulated and aggregated (Figure 3). In addition, expression of crdR was higher in the stationary phase than in the exponential phase, and crdR expression further activated curdlan biosynthesis in the ATCC31749 strain to generate a biofilm. This suggests that curdlan may be critical for biofilm formation in ATCC31749 for improving stress tolerance to harsh conditions.
Bioinformatic analysis indicated that crdR can be grouped into a conserved XRE-family of transcriptional factors that is comprised of HipB in E. coli, PhrR in S. meliloti, CH00371 in R. etli and PraR in A. caulinodans (Additional file 1) . Apparently, diverse stress can induce expressions of XRE-family transcriptional factors. Combining with HipA, HipB, a crdR homologue of E. coli that mediates multidrug stress tolerance can bind to its cis elements with conserved sequences of TATCCN 8 GGATA (where N8 indicates any 8 nucleotides). Genomic scanning indicated that there is no HipA counterpart in the ATCC31749 strain, and that there were no conservative TATCCN8GGATA sequences in the promoter region of the crd operon. However, crdP does have three distinct hairpin structures located at the −10, −35, and −92 regions (Additional file 2), which are putative crdR binding sites. That purified crdR can bind to an amplified fragment containing the −92 region of crdP more than the −53, and −13 regions indicates that those region are likely the crdR binding site. HipAB is a heterodimer of a transcriptional repressive regulator , and crdR may play a different role as a transcriptional activator in the form of a homotetramer or homodimer, which must be confirmed with additional studies. PhrR in S. meliloti was affected by low pH, Cu2 +, Zn2 + and H2O2 stresses . Expression of CH00371 in R. etli was promoted by oxidation and osmotic shock [29,30]; whereas expression of PraR in A. caulinodans was increased by low nitrogen . Primary data from transcriptome analysis obtained from RNAseq indicates that expression of crdR in the curdlan fermenting phase with both low pH and limited nitrogen was twice as great as that in the growing phase of bacterium in LB medium (data not shown) and q-RT-PCR analysis of crdR expression agreed with RNA-Seq data (Table 3). Thus, crdR expression should be triggered by stress factors as well.
Most organisms within the genera of Rhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, and Sinorhizobium are symbiotic bacteria to various leguminous plants [32,33]. After bacteria enter plant tissues, their environment changes to low pH (normally 5.5), with limited nitrogen and sufficient carbohydrates [32,33], and this environmental shift affects strain morphology and physiology. To survive with limited nitrogen, genes related to bacterial nitrogen fixing and those for low pH tolerance are expressed. Transcriptional factors such as PhrR and its homologues are instrumental for expression of low pH tolerance genes. Also, with abundant carbohydrates from host photosynthesis, bacterial extracellular polysaccharides (EPS) are synthesized to produce a biofilm  to protect the bacteria from stress. Also, the biofilm provides a low-oxygen environment inside the bacteria to support nitrogen fixing reactions. This concept is in agreement with research with PraR from A. caulinodans ORS571, a homolog of crdR, which can mediate stem nodule formation, regulate expression of Reb genes, and increase nitrogen fixation of bacterial strains within stem nodules . Therefore, biosynthesis of curdlan regulated by crdR may originate from ancestral characteristics for survival within a host plant. Currently, detailed regulatory mechanisms of crdR expression, controlled by disadvantageous conditions such as low pH and limited nitrogen remain unknown. However, reports regarding stringent response signal(p)ppGpp, which can induce EPS biosynthesis and bacterial biofilm formation  is worthy of study. We hypothesize that crdR expression may be activated by (p)ppGpp, which accumulates under stressful conditions.
In this study, we confirmed that crdR regulates curdlan synthesis by activating expressions of its biosynthetic genes. Ours is the first work to identify XER family transcriptional factor which can activate EPS biosynthesis. crdR may be a multiple-effect regulator controlling expression(s) of the curdlan synthesis gene(s) in ATCC31749 under oxidative stress/low pH and/or limited nitrogen with abundant sugar. This function of curdlan regulation indicates that curdlan biosynthesis of ATCC31749 under harsh conditions may have evolutionary origin.
The study was supported by the Chinese Natural Science Foundation (No. 31170057) and the Yunnan Natural Science Foundation (No. 2010CD054).
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