Exopolysaccharide Yield and Environmental Adaptation of Rhizobia Regulated by Gene envZ, iscS and asnC

doi:10.13560/j.cnki.biotech.bull.1985.2024-0388

Abstract

Abstract:

【Objective】 Rhizobium yanglingense CCBAU 01603 form efficient nitrogen-fixing nodules with Caragana legumes. Alk_40, one of the offspring of CCBAU 01603, was obtained through experimental evolution in alkaline medium for 500 generations. It was discovered that a significant decrease in exopolysaccharide（EPS）yields of Alk_40 with specific SNPs changes in the gene envZ, iscS, and asnC. EPS yields and environmental adaptability of rhizobia regulated by these key genes were investigated. 【Method】 Three mutants of rhizobia were created by deleting gene envZ, iscS, and asnC, respectively. EPS yields and growth under different environmental conditions of mutants were assessed. 【Result】 In comparison to CCBAU 01603, the envZ, iscS, and asnC mutants presented significantly lower EPS yields in YMA medium, demonstrated diminished fitness in acidic environments, and had a weakened tolerance to high temperatures of 65℃. This observation suggests a correlation between the EPS yield of rhizobia and their resistances to acidic or heat stress. In addition, the mutants lacking the envZ and asnC genes showed the sensitivity to NaCl, resulting in severely limited growth and reproduction in NaCl-containing media. Furthermore, their adaption in alkalescent environments significantly reduced as well. 【Conclusion】 It is discovered that EPS yields of rhizobia can be regulated by gene envZ, iscS, and asnC.

Key words: Rhizobium, envZ, iscS, asnC, exopolysaccharide, environmental adaptation

YANG Bing-jie, YUAN Xiao-xia, GAO Meng-zhe, SHEN Ao-long, LI Hua, JI Zhao-jun. Exopolysaccharide Yield and Environmental Adaptation of Rhizobia Regulated by Gene envZ, iscS and asnC[J]. Biotechnology Bulletin, 2024, 40(11): 277-284.

Figures/Tables 8

Table 1 Strains in this study

菌株 Strain	特征 Description	来源 Source
R. yanglingense CCBAU 01603	野生型菌株 Wild-type strain NA^r	实验室保藏 Preserved in Lab
Alk_40	CCBAU 01603的进化菌株 Evolved clone of CCBAU 01603	实验室保藏 Preserved in Lab
ΔenvZ	envZ基因敲除突变株 Gene envZ deletion mutant NA^r	已构建 Established in Lab
ΔiscS	iscS基因敲除突变株 Gene iscS deletion mutant NA^r	已构建 Established in Lab
ΔasnC	asnC基因敲除突变株 Gene asnC deletion mutant NA^r	已构建 Established in Lab

Fig. 1 Comparative analysis of key gene sequences and translated amino acid sequences of R. yanglingense CCBAU01603 and evolved Alk_40 A is from the comparison of the envZ gene sequence and amino acid composition; B is from the comparison of the iscS gene sequence and amino acid composition; C is from the comparison of the asnC gene sequence and amino acid composition

Fig. 2 Growth of each rhizobia on YMA solid medium

Fig. 3 Exopolysaccharide yield among different rhizobia *: 0.01<P≤0.05; **: 0.001<P≤0.01; ***: 0.0001<P≤0.001; ****: P ≤0.0001, the same below

Fig. 4 Growth of rhizobia in the media with different concentrations of NaCl A: Liquid medium containing 0.1% NaCl. B: Liquid medium containing 0.2% NaCl. C: Liquid medium containing 0.3% NaCl. D: Liquid medium containing 0.4% NaCl

Table 2 Correlation between exopolysaccharide yield and OD600 values

环境条件Environmental conditions		皮尔逊相关系数Pearson’s correlation coefficient	显著性（双尾） Sig（2-tailed）（P）
盐浓度NaCl concentrations	0.1%	0.734ns	0.158
	0.2%	0.613ns	0.271
	0.3%	0.508ns	0.382
	0.4%	-0.211ns	0.733
pH 值 pH Value	4.02	0.981**	0.003
	5.01	-0.087ns	0.89
	8.99	0.389ns	0.517
	9.98	0.138ns	0.825
热处理温度Heat treatment temperature	28℃	0.976**	0.004
	37℃	0.840ns	0.075
	65℃	0.979^**	0.004

Fig. 5 Growth of rhizobia in the medium with different pH A: Liquid medium with pH=4.02; B: liquid medium with pH=5.01; C: liquid medium with pH=6.99; D: liquid medium with pH=8.99; E: liquid medium with pH=9.98

Fig. 6 Growth of rhizobia after treatment at different temperature A: Negative control group treated at 28℃ for 10 min; B: treated at 37℃ for 10 min; C: treated at 65℃ for 10 min

References 31

[1]	Ji ZJ, Yan H, Cui QG, et al. Competition between rhizobia under different environmental conditions affects the nodulation of a legume[J]. Syst Appl Microbiol, 2017, 40(2): 114-119.
[2]	Ji ZJ, Wu ZY, Chen WF, et al. Physiological and symbiotic variation of a long-term evolved Rhizobium strain under alkaline condition[J]. Syst Appl Microbiol, 2020, 43(5): 126125.
[3]	Hollingsworth RI, Dazzo FB, Hallenga K, et al. The complete structure of the trifoliin A lectin-binding capsular polysaccharide of Rhizobium trifolii 843[J]. Carbohydr Res, 1988, 172(1): 97-112.
[4]	Palhares Farias T, de Melo Castro E, Marucci Pereira Tangerina M, et al. Rhizobia exopolysaccharides: promising biopolymers for use in the formulation of plant inoculants[J]. Braz J Microbiol, 2022, 53(4): 1843-1856.
[5]	Park S, Shin Y, Jung S. Structural, rheological properties and antioxidant activities analysis of the exopolysaccharide produced by Rhizobium leguminosarum bv. viciae VF₃9[J]. Int J Biol Macromol, 2024, 257(Pt 2): 128811.
[6]	Muszynski A, Laus M, Kijne JW, et al. Structures of the lipopolysaccharides from Rhizobium leguminosarum RBL5523 and its UDP-glucose dehydrogenase mutant(exo5)[J]. Glycobiology, 2011, 21(1): 55-68.
[7]	Janczarek M, Rachwał K, Kopcińska J. Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover[J]. Plant Soil, 2015, 396(1): 257-275.
[8]	Janczarek M. The ros/MucR zinc-finger protein family in bacteria: structure and functions[J]. Int J Mol Sci, 2022, 23(24): 15536.
[9]	Kenney LJ, Anand GS. EnvZ/OmpR two-component signaling: an archetype system that can function noncanonically[J]. EcoSal Plus, 2020, 9(1): 10.1128/ecosalplus.ESP-10.1128/ecosalplus0001-2019.
[10]	Fu DD, Wu JM, Wu XY, et al. The two-component system histidine kinase EnvZ contributes to Avian pathogenic Escherichia coli pathogenicity by regulating biofilm formation and stress responses[J]. Poult Sci, 2023, 102(2): 102388.
[11]	Das M, Sreedharan S, Shee S, et al. Cysteine desulfurase(IscS)-mediated fine-tuning of bioenergetics and SUF expression prevents Mycobacterium tuberculosis hypervirulence[J]. Sci Adv, 2023, 9(50): eadh2858.
[12]	Yan SQ, Zhen JF, Li YZ, et al. Mycobacterium Lrp/AsnC family transcriptional factor modulates the arginase pathway as both a sensor and a transcriptional repressor[J]. J Genet Genomics, 2021, 48(11): 1020-1031.
[13]	刘景煜, 李晨, 肖林刚, 等. 双水相萃取法分离纯化金针菇子实体多糖[J]. 食品与发酵工业, 2017, 43(5): 255-260.
	Liu JY, Li C, Xiao LG, et al. Isolation and purification of aqueous two-phase extraction of polysaccharides from Flammulina velutipes[J]. Food Ferment Ind, 2017, 43(5): 255-260.
[14]	Lee D, Lee YM, Hye Shin S, et al. A simple protein histidine kinase activity assay for high-throughput inhibitor screening[J]. Bioorg Chem, 2023, 130: 106232.
[15]	Park H, Saha SK, Inouye M. Two-domain reconstitution of a functional protein histidine kinase[J]. Proc Natl Acad Sci USA, 1998, 95(12): 6728-6732.
[16]	Tomomori C, Tanaka T, Dutta R, et al. Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ[J]. Nat Struct Biol, 1999, 6(8): 729-734.
[17]	Lill R, Freibert SA. Mechanisms of mitochondrial iron-sulfur protein biogenesis[J]. Annu Rev Biochem, 2020, 89: 471-499.
[18]	Fujishiro T, Nakamura R, Kunichika K, et al. Structural diversity of cysteine desulfurases involved in iron-sulfur cluster biosynthesis[J]. Biophys Physicobiol, 2022, 19: 1-18.
[19]	Nogales J, Campos R, BenAbdelkhalek H, et al. Rhizobium tropici genes involved in free-living salt tolerance are required for the establishment of efficient nitrogen-fixing symbiosis with Phaseolus vulgaris[J]. Mol Plant Microbe Interact, 2002, 15(3): 225-232.
[20]	Modrzejewska M, Kawalek A, Bartosik AA. The lrp/AsnC-type regulator PA2577 controls the EamA-like transporter gene PA2576 in Pseudomonas aeruginosa[J]. Int J Mol Sci, 2021, 22(24): 13340.
[21]	Thaw P, Sedelnikova SE, Muranova T, et al. Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family[J]. Nucleic Acids Res, 2006, 34(5): 1439-1449.
[22]	Acosta-Jurado S, Fuentes-Romero F, Ruiz-Sainz JE, et al. Rhizobial exopolysaccharides: genetic regulation of their synthesis and relevance in symbiosis with legumes[J]. Int J Mol Sci, 2021, 22(12): 6233.
[23]	Laus MC, Logman TJ, Van Brussel AAN, et al. Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra[J]. J Bacteriol, 2004, 186(19): 6617-6625.
[24]	Sánchez-Andújar B, Coronado C, Philip-Hollingsworth S, et al. Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii[J]. Mol Gen Genet, 1997, 255(2): 131-140.
[25]	Marczak M, Żebracki K, Koper P, et al. A new face of the old gene: deletion of the PssA, encoding monotopic inner membrane phosphoglycosyl transferase in Rhizobium leguminosarum, leads to diverse phenotypes that could be attributable to downstream effects of the lack of exopolysaccharide[J]. Int J Mol Sci, 2023, 24(2): 1035.
[26]	Wisniewski-Dyé F, Downie JA. Quorum-sensing in Rhizobium[J]. Antonie Van Leeuwenhoek, 2002, 81(1-4): 397-407.
[27]	Ji YY, Zhang BL, Zhang P, et al. Rhizobial migration toward roots mediated by FadL-ExoFQP modulation of extracellular long-chain AHLs[J]. ISME J, 2023, 17(3): 417-431.
[28]	Hoang HH, Becker A, González JE. The LuxR homolog ExpR, in combination with the Sin quorum sensing system, plays a central role in Sinorhizobium meliloti gene expression[J]. J Bacteriol, 2004, 186(16): 5460-5472.
[29]	Bustamante JA, Ceron JS, Gao IT, et al. A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts[J]. PLoS Genet, 2023, 19(10): e1010776.
[30]	Gao MJ, Liu ZL, Zhao ZS, et al. Exopolysaccharide synthesis repressor genes(exoR and exoX)related to curdlan biosynthesis by Agrobacterium sp[J]. Int J Biol Macromol, 2022, 205: 193-202.
[31]	Morcillo RJL, Manzanera M. The effects of plant-associated bacterial exopolysaccharides on plant abiotic stress tolerance[J]. Metabolites, 2021, 11(6): 337.