The Mechanism Of Global Regulator CodY In Streptococcus Thermophilus

Streptococcus thermophilus is a major dairy starter traditionally used in combination with Lactobacillus delbrueckii subsp. bulgaricus or Lb. helveticus for the manufacture of yogurt and cheeses. S. thermophilus is a "generally recognised as safe"（GRAS） species and over1021live cells are ingested annually. Notably, S. thermophilus forms a unique carbon metabolic system to efficiently utilize the lactose in milk to provide energy for tis growth. S. thermophilus is a fastidious microorganism and requires an exogenous source of amino acids or peptides for optimal growth. As milk is poor in these low-molecular-weight compounds, its growth largely depends on proteolytic system to achieve hydrolysis of caseins. The weak activity or missing of cell envelope proteinase strongly limits the growth. S. thermophilus acquries a well-developed nitrogen pathway for de novo biosynthesis of amino acids by horizontal gene transfer （HGT） due to its adaptation to milk. During dairy processes, S. thermophilus always exposes to reactive O2species （ROS）, heat shock, phage infection stresses, and their potential deleterious effects on growth, fermentative capabilities and viability, consequently have repercussions on texture and flavour of the final products. Fortunately, all bacteria have additional layers of control that coordinate the use of nutrients and the resistance to environmental stress using global regulators. CodY is a highly conserved protein in the low-G-C group Gram-positive bacteria, defines a unique family of regulatory proteins. CodY is helpful for bacteria to adaption to poor nutritional availability, and also controls the catabolic pathways, environment response as well as the avirulence of strain. The effects of CodY is stimulated by interaction with either of two ligands, GTP or BCAA. However, the mechanism of CodY regulation possesses strain specificity.Analysis of the annotated S. thermophilus genome sequences identified a conserved gene designated codY. Here, we focused on the global regulator CodY, and studied the metabolic regulation and stress response of S. thermophilus ST2017. The carbon and nitrogen metabolism regulatory network was elucidated by transcriptome analysis, the regulatory mechanism of CodY was investigated by electrophoretic mobility shift assay （EMSA） and isothermal titration calorimetry （ITC）, and clarified the role of CodY in stress response. The detailed contents and results of this thesis were as follows:The CodY regulon of S. thermophilus ST2017To identify genes regulated by CodY, the codY gene of S. thermophilus was knockout using the thermosensitive plasmid pG+host9, and generating the mutant ST2017△codY. To establish the extent of the CodY regulon in S. thermophilus ST2017, RNA-seq was undertaken. The transcriptome analysis released87differently expressed genes in ST2017△codY, and62out of87genes were repressed by CodY. These genes were divided into different groups based on the function of regulated genes as follows:（1） Amino acids biosynthesis and transport. The genes involved in biosynthesis of BCAA, tryptophan, cysteine, aspartic acid, serine and threonine synthesis were repressed. The guanosine hydratase gene （hutU）, during the histidine degradation process is the sole gene actived by CodY in amino acids metabolism.（2） The global regultor CodY aslo controlled the glyceraldehyde3-phosphate dehydrogenase which is involved in EMP pathway and phosphate mutase which is involved in Leloir pathway.（3）The gdhA gene was significantly derepressed in codY mutant.（4） A number of genes, such as STND₀202-0203、STND₁235-1232and STND₀733involved in ABC transporter and ion channel were derepressed.（5） Transcriptome data showed that CodY also involved in regulation of genes respond to environmental stresses. Chaperone DnaK and GroESL operons in codY mutant were significantly upregulated. Osmotic stress-related STND₀113, STND₀135, and oxidative stress-related STND₁346were downregulated.（6）Meanwhile the two-component system TCS01involved in environmental stress response is also regulated by CodY.（7） CodY actived the transcription of hypoxanthine guanine （IMP） de novo synthesis operon, while the transcription of uracil synthesis operon was repressed.（8） A number of hypothetical proteins were regulated by CodY. In conclusion, the global regulator CodY were involved in the regulation of varieties of metabolic pathways, especially in the fine-turning of nitrogen metabolism. Meanwhile, CodY also regulated the genes responding to environmental stress.The regulation mechanism of CodY in S. thermophilus ST2017In Gram-positive bacteria, the regulation CodY binds the DNA fragment with the help of the affecters GTP and BCAA. To find out the affecter CodY needed in S. thermophilus ST2017, the promoter of livJHMG operon identified by transcriptome analysis was PCR amplified, and the PCR product was mixed with purified CodY to perform out the EMSA analysis. This binding ability of CodY was enhanced by addition of15mM BCAA, do not of GTP. The EMSA expriments showed that the S. thermophilus CodY responded to the intracellular BCAA concentrations but not to physiological fluctuations in intracellular GTP. To further confirm the positive effect of BCAA, the livJHMG promoter Pliv was fused with green fluorescent protein gene （gfp） and the plasmid pSEC-Pliv-gfp was expressed in S. thermophilus ST2017. As a result, the level of fluorescence intensity was reduced by addition of BCAA, and this confirmed the positive effect of BCAA in vivo. A15-bp conserved motif A[AT]T [AT]TTC[TC] GA[ACT][AT]TT in the promoter regions of CodY regulated genes was identified using MEME soft. Furthermore, there was a high similarity to the canonical CodY-box, AATTTTCWGAAAATT, which was previously described based on analysis of L. lactis and B. subtilis CodY-regulated genes. The interaction of CodY and DNA containing the15-bp conserved motif was monitored by ITC analysis. The results proved that CodY and conserved motif had a significant combination effect, indicating that the conserved sequence has a crucial role in the identification of CodY. BCAA could effectively improve the affinity, and the dissociation constant Kd reduced from780nM to8.33nM.The global regulator CodY coordinates the flow of carbon and nitrogen metabolismS. thermophilus has a complete metabolic pathway from pyruvate to a-oxoglutarate, and the metabolite a-oxoglutarate stands at the crossroads between carbon metabolism and nitrogen metabolism. As one of the substrates of glutamate dehydrogenase （gdhA）, a-oxoglutarate provides the de novo carbon skeleton for glutamate. Moreover, as the product of glutamate metabolism by glutamate dehydrogenase, a-oxoglutarate can be the entry point into central metabolism for the carbon skeletons of several amino acids. The transcriptomics and real-time PCR showed that glutamate dehydrogenase gene （gdhA） was significantly repressed by CodY. EMSA further confirmed that CodY had a direct role in the regulation of gdhA transcription. The glutamate dehydrogenase enzyme activity of S. thermophilus ST2017was190U, and deletion of codY increased the enzyme activity to330U, while overexpression of codY in S. thermophilus ST2017△codY decreased the enzyme activity to110U. The results showed that CodY directly inhibited the expression of gdhA. In S. thermophilus ST2017, the glutamate dehydrogenase catalyzed both a-ketoglutarate synthesis to glutamic acid with a Km value of0.131±0.030mM, and glutamic acid degradation to a-ketoglutarate with a Km value of1.170±0.088mM. The Km value of a-oxoglutarate to glutamate synthesis was much lower than that of glutamate to a-oxoglutarate. This metabolic advange forced the carbon into nitrogen metabolism, and effectively utilized the lactose in milk. Histidine, cysteine, proline and methionine were added to Chemical-defined medium （CDM） to maintain the growth of S. thermophilus ST2017. When the lactose concentration in CDM increased from0.5%to1.0%, the biomass increased from0.768±0.011to1.048±0.061in S. thermophilus ST2017, while the biomass of ST2017△codY rised from0.822±0.017to1.381±0.023. We supposed that the increased biomass was from the conversion of lactose to amino acids, under nitrogen limiting condition. To verify the role of glutamate dehydrogenase in the conversion of lactose to amino acids, the gdhA gene was deleted in S. thermophilus ST2017and ST2017△codY, yielding ST2017△gdhA and ST2017△codY△gdhA, and the biomass decreased to0.305±0.034and0.531±0.041in CDM with0.5%lactose. The biomass only slightly increased to0.328±0.009and0.581±0.104with the increase of lactose to1.0%. The results showed that, under nitrogen limiting condition, S. thermophilus can turn carbon source into amino acids for cell growth through glutamate dehydrogenase, the deletion of codY increased this transformation capability. When the link between carbon and nitrogen metabolism was blocked（inactive gdhA gene）, the biomass was severely decreased.In carbon limiting condition, the biomass of S. thermophilus ST2017and ST2017△codY increased to1.340±0.067and1.403±0.053when the nitrogen （Tryptone） increased from0.25%to0.5%, while the biomass of ST2017△gdhA and ST2017△codY△gdhA was only increased to1.188±0.004and1.164±0.044. This result confirmed that the capacity of converting carbon to nitrogen was also reduced in AgdhA mutant, and eventually limit the cell growth. This chapter elaborated on the interconversion of carbon, nitrogen metabolism in S. thermophilus by global regulator CodY.The role of CodY in response to environmental stressThe ability of S. thermophilus to cope with the environmental stresses during manufacture and preservation （oxidative stress, high temperature, and phage infection） is essential for its performance as a starter. This research found that S. thermophilus ST2017global regulator CodY responded to environmental stress by controlling the two-component system TCS01, while CodY itself can regulate various stress response proteins. The results showed that CodY directly regulated the transcription of DnaK, GroESL, STND₀113and STND₀135, while the transcription of STND₁346gene was regulated by the two-component system TCS01directly. S. thermophilus is a facultative anaerobe, and reduces the intracellular concentration of superoxide radicals and oxygen molecules by superoxide dismutase and NADH oxidase. The glutathione （GSH/Gr/Grx） system is responsible for the bacterial intracellular low redox potential and the maintenance of proteins in their reduced state. To enhance bacterial resistance to reactive oxygen, the glutathione synthetase gene （STND₁346） was derepressed in ST2017△coY, while the glutathione synthetase gene（STND₁346） was directly controlled by the two-component system TCS01. The global regulator CodY controlled the expression of STND₁346indirectly by repressing the two-component system TCS01. Comparing oxidative stress tolerance of S. thermophilus ST2017and ST2017△codY After H2O2treatment, the survival of ST2017is more than3-fold than that of ST2017△codY in CDM containing glutamate and glycine, and the concentration of glutathione was3.40±0.28μM in ST2017and2.47±0.04μM in ST2017△codY. The survival of ST2017and ST2017△codY was significantly decreased in CDM without glutamate and glycine, and the concentration of glutathione was basically undetectable. The results showed that, the susceptible to oxidative stress of ST2017△codY compared to ST2017was due to the decrease of glutathione concentration. Further research showed that, the upregulation of DnaK and GroESL in codY mutant significantly improved the heat shock tolerance, while the downregulation of STND₀113and STND₀135genes reduced the osmotic stress resistance. These results showed that the global regulator CodY was responded to oxidative, temperature, osmotic stress, and had an important regulatory function in S. thermophilus ST2017.The systematic use of the same S. thermophilus strains in dairy processes has been impaired by the ubiquitous presence of virulent phages. This research found that the cas5gene in S. thermophilus CRISPR1/Cas system was strongly repressed by the global regulator CodY and two-component system TCS01directly. The over-expression of cas5gene greatly increased the loss rate of exogenous plasmid in S. thermophilus ST2017, while increasing bacterial resistance to phage. Further studies showed that no new spacer insertion was found in CRISPR sequences, this was different from the mechanism of type Ⅱ CRISPR/Cas systems. Furthermore, we found that Cas5protein had a tracrRNA and crRNA independent nuclease activity in vitro assay, and could cut the plasmid and λ-DNA with a non-specific manner. We speculated that the nuclease activity of Cas5protein increased the loss rate of exogenous plasmid, and mad it easier to generate phage immunity. This was the first evidence of S. thermophilus CRISPR/Cas system to immunize exogenous DNA by non-specific nuclease activity of Cas5.Construction and application of food-grade expression system for lactis acid bacteria based on alanine racemase geneLactic acid bacteria （LAB） are widely used as probiotics and have great potential to serve as antigen delivery vehicle for oral vaccines. However, the application of genetically modified organisms in food products and practical vaccines requires safe and sustainable genetic tools devoid of any antibiotic-resistance markers. Therefore, sophisticated food-grade marker systems are being developed. Here, we focused on the air gene as a selection marker, and an inducible food-grade system as well as a constitutive food-grade system were constructed with DNA fragments entirely from lactic acid bacteria. The new inducible food-grade expression system in L. lactis with pSEC replicon, PnisA promoter and air selection marker was constructed. The green fluorescence protein （gfp） was used as a reporter for gene expression to verify the feasibility of the new host/vector system. We, therefore, focused on the lactococcal strain as a cell factory, which was used to express high amount of the capsid protein of porcine circovirus type Ⅱ（dCap）. The maximum amount of dCap reached1,059±99μg/1, and after a2h nisin pulse induction, the dCap protein maintained secretion even10h after the nisin pulse, and decreased to940±101μg l-1. The results verified the potential application of the new inducible food-grade system for an oral vaccine purpose. Enterotoxigenic Escherichia coli （ETEC） strains are the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factors in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Considering of the safety of the genetic modified LAB, a constitutive food-grade secretion system was constructed with promoter of S-layer protein, replicon and selection marker alanine racemase（alr）. To evaluate the feasibility of the system, Nuclease （NucB） from Staphylococcus aureus was used as a reporter gene to express in both L. lactis and Lb. casei. Subsequently, extracellular expression of fimbrial adhesin FaeG of ETEC was confirmed by western blot analysis. These two new systems were real food-grade system as appropriate antigen delivery systems, and also suitable for the production of ingredients in food industry.