金黄色葡萄球菌耐热核酸酶的功能鉴定及表达调控
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摘要
金黄色葡萄球菌（Staphylococcus aureus）是引起人类多种感染性疾病及食物中毒的重要病原菌之一，它导致的食物中毒和医院感染已经成为世界范围内影响公共卫生与健康的重要问题。耐热核酸酶是金黄色葡萄球菌重要的毒力因子之一，同时也是分离鉴定金黄色葡萄球菌的重要判定指标。课题组前期研究发现,一个新的编码耐热核酸酶的基因nuc2和已知功能的耐热核酸酶基因nuc1同时存在于基因组中，并能表达耐热核酸酶的活性。本论文从nuc2耐热核酸酶结构和特性的比较入手，围绕新的耐热核酸酶nuc2基因的功能鉴定展开研究，进而以基因表达、功能鉴定、调控规律为重点比对分析金黄色葡萄球菌两种耐热核酸酶的差异，主要研究内容和结果如下：
1.采用生物信息学分析方法对金黄色葡萄球菌基因组中的nuc1、nuc2基因编码的蛋白质序列及结构进行比对，构建了不同种属间Nuc蛋白的进化树，揭示了金黄色葡萄球菌两个核酸酶在进化上的差异性。结果表明，Nuc2是一个在葡萄球菌属内进化较为保守的蛋白，而Nuc1则可能起源于其它菌属的水平转移。此外，在大肠杆菌中成功表达了两种核酸酶，对比研究了它们在体外表达时的酶活性特征，揭示了金黄色葡萄球菌Nuc2的酶学特征：最适反应温度为50℃，最适pH值为10，是一种非特异核糖核酸酶，适当浓度的Ca~(2+)、Mg~(2+)、DTT、β-mecaptoethanol、TritonX-100、Tween-20和Urea都能够提高Nuc2的酶活性，而Zn~(2+)、Mn~(2+)、EDTA和SDS则是Nuc2酶活性抑制剂。和Nuc1相比，体外表达的Nuc2酶活性较低，且对高温和重金属离子的耐受力明显低于Nuc1，这和Nuc2蛋白结构上不稳定的推测结果相符。本实验从序列、结构到酶活性特征上很好地区分了金黄色葡萄球菌两个重要的耐热核酸酶，对新近发现的耐热核酸酶Nuc2的特征作了较为详尽的研究，为其功能应用奠定了基础。
2.采用Taqman荧光定量PCR方法比较了两个耐热核酸酶基因在转录水平上的差异。通过对金黄色葡萄球菌不同生长阶段nuc1和nuc2基因mRNA表达水平的比较分析，发现nuc1和nuc2基因在生长过程中的表达模式并不相同：nuc1基因的表达在对数后期达到最大值，和耐热核酸酶活性测定结果一致；nuc2基因则在生长初期达到最大值，到对数后期反而降低。另外，通过测定金黄色葡萄球菌双组分调节子sae的缺失突变对nuc1和nuc2基因表达的影响，结果发现，sae基因缺失显著下调nuc1基因的表达；而nuc2基因在sae缺失突变株中表达几乎不受影响。金黄色葡萄球菌临床分离株中的nuc1基因表达变化差异明显，nuc2基因的表达则相对稳定。通过研究nuc1和nuc2基因在表达上的相关性，发现nuc1和nuc2基因受到不同的调控机制调控，且nuc1基因的表达是决定耐热核酸酶活性表型的主要因素。
3.利用微生物的同源重组双交换的遗传原理，成功构建了耐热核酸酶nuc2基因单缺失突变株和nuc1、nuc2基因双缺失突变株，并在分子水平进行了验证。突变株耐热核酸酶活性的测定结果表明，nuc2基因缺失后，酶活性比野生型降低，双突变株则检测不到耐热核酸酶活性。构建互补载体转化双突变株,互补菌株的酶活性测定结果发现，nuc1基因互补双缺失突变株能使其完全恢复到野生型的酶活水平，nuc2基因的互补只能恢复菌株nuc1基因突变后Nuc2的酶活表型。基因突变和互补实验确定了nuc2基因在金黄色葡萄球菌中具备耐热核酸酶活性表达功能，也进一步证实了nuc1基因在耐热核酸酶活性表型中占有主导地位的结论。
4.利用基因表达谱芯片技术研究了两种核酸酶基因突变后对金黄色葡萄球菌RN4220转录组的影响，采用连锁转录及基因代谢途径定位的新分析方法，对两种核酸酶基因突变所影响的差异基因进行分析，结果表明，nuc1基因缺失显著影响了393个基因转录水平的变化，而nuc2的缺失显著影响了89个基因转录水平的变化。虽然两组基因都涉及到菌株的氨基酸代谢、糖代谢、复制修复、核酸代谢及调控因子，但在相同位置发生差异变化的基因只有20个，且调控水平都不相同。挑选21个差异基因，采用荧光定量PCR验证了芯片结果的可靠性。值得注意的是，△nuc2缺失突变株和精氨酸脱亚胺酶途径相关的基因座均下调，和ABC转运子相关的基因座上调，连锁调控的代谢途径还涉及到嘌呤代谢、甘氨酸、苏氨酸、异亮氨酸等氨基酸代谢途径。△nuc1基因缺失主要下调了和磷酸转移酶系统、脂代谢及毒力调控因子的连锁基因的表达，上调了尿素酶基因及蛋白酶基因。△nuc1和△nuc2缺失突变株在转录水平分别调控了不同的基因表达，且各有分工。
5.为了更全面了解两种核酸酶基因的突变对于细胞代谢功能的影响，利用高通量的表型分析新技术--表型芯片(PM)研究了金黄色葡萄球菌两种耐热核酸酶突变株和亲本菌株RN4220的1100多种不同的表型，包括各种碳源、氮源、磷源、硫源的利用，对氨基酸及其他营养物质的代谢、渗透特征、pH及抗生素敏感性测试。和金葡亲本株相比，Δnuc2突变株的PM结果有13种表型减弱了1.5倍以上，包括对3种碳、硫源的利用，对7种氮源的利用，涉及氨基酸短肽及氨的代谢。突变株全部下调的表型说明了nuc2基因对金黄色葡萄球菌细胞的生理代谢的正常进行起着重要的作用； Δnuc1突变株的差异表型有9个，涉及氮源和磷源的代谢以及6个在酸性pH下的表型变化，和nuc2突变株表型不同的是，Δnuc1突变株中7种表型变化都是增强的，尤其是在酸性pH时突变株的生长和对氨基酸的利用上。25种抗生素敏感性测定结果发现，Δnuc2突变株和Δnuc1突变株对2-4种头孢类抗生素的抗性有所增加，这可能与膜蛋白的表达差异相关。生长曲线的测定结果表明，在酸性pH5条件下Δnuc1突变株的生长的明显高于Δnuc2菌株和野生型，且菌膜形成量明显增多，这与Δnuc1突变株在酸性条件下代谢表型的增强有重要的联系。
总之，本论文揭示了耐热核酸酶基因nuc2的特征及功能，对两个耐热核酸酶在金黄色葡萄球菌中表达调控机制进行了探索，为更好地了解金黄色葡萄球菌耐热核酸酶的作用机制具有重要意义。
Staphylococcus aureus is one of the most important pathogens thatcan cause food-borne disease, as well as nosocomial infections.Staphylococcal food poisoning and nosocomial infections are of majorconcerns in public health worldwide. S. aureus produces thermonucleasewhich is known as one of the most important virulence factors.Thermonuclease is widely used as a unique marker for detection andidentification of S. aureus. Our previous studies have revealed theexistence of two functional thermostable nucleases encoded by twodifferent genes (nuc1and nuc2) in the S. aureus genome. To characterizethe function of Nuc2enzyme, our research started from comparisonanalysis of Nuc2and Nuc1on the structure and feature. Furthermore, thefunctional and regulatory differences of the two thermonucleases in S.aureus were also charaterized. The main findings and results are listed asfollows:
1. Sequence alignments and phylogenetic analysis were carried outto study the evolution of Nuc1and Nuc2using bioinformatics tools. Theresults revealed that Nuc2protein was more conserved in thestaphylococci group compared with Nuc1protein, while the nuc1genemight have been acquired by lateral transfer from another pathogenicspecies. Two recombinant thermonucleases were successfully expressedin Escherichia coli, and their nuclease activities were further characterized and compared. The results showed that the optimal reactiontemperature and pH for recombinant Nuc2were50℃and pH10,respectively, and it was a sugar non-specific nucleases. The enzymaticactivity of Nuc2was stimulated in the presence of Ca~(2+), Mg~(2+),dithiothreitol, β-mecaptoethanol, TritonX-100, Tween-20, and urea;however, the activity decreased dramatically when exposed to heavymetals such as Zn~(2+)and Mn~(2+), and in the presence of EDTA or SDS.Nuc2showed a weaker activity, lower thermostability and differentsensitivity to the compounds compared with Nuc1, which showed acorrelation with the lack of a consensus pattern and changes of thehydrophobic core residues in the structure of Nuc2as predicted. Thisstudy determined important characteristics of the novel thermonuclease(Nuc2) in S. aureus and provided a discriminatory picture in therelationship on evolutionary and enzymatic differences of Nuc1and Nuc2by comparative analysis. This information helped us to better understandthe gene function and the potential application of this newthermonuclease gene.
2. To study the expression of these two genes, comparative mRNAanalysis of nuc1and nuc2was carried out by Taqman quantitative real-time PCR. Distinct expression patterns were observed at different growthstages. The maximum level of nuc1transcripts appeared at the post-exponential growth phase, and the transcription level correlated well withthermonuclease activity results. In contrast, nuc2transcripts were mostlyexpressed at the early exponential phase and declined at the post-exponential growth phase. Effects of sae mutation on the expression ofthermostable nuclease genes were also determined. The nuc1geneexpression was notably down-regulated in a sae mutant and the nuc2genewas barely changed in the sae mutant. Furthermore, unlike the expressionof nuc1that varied in three different S. aureus clinical strains, the transcription of nuc2remained relatively constant. Comparativeexpression analysis of two thermostable nuclease genes showed that nuc1and nuc2gene were not co-regulated in Staphylococcus aureus, and nuc1expression plays a more pronounced role in thermonuclease activitiesthan that of nuc2.
3. The nuc2knock out mutant and the nuc1and nuc2double deletionmutant of S. aureus were successfully constructed by homologousrecombination, and the mutants were confirmed on the molecular level.Thermonuclease assays revealed functional activity differences of nuc1and nuc2in S. aureus. Compared to the parental strain, RNΔnuc1andRNΔnuc2exhibited reduced activity zones on the plate. Thethermonuclease activity of the RNΔnuc2was higher than that of theRNΔnuc1on the toluidine blue-DNA agar. No activity was observed inthe nuc1and nuc2double deletion mutant. Complementation plasmidswere constructed and the complementation of the nuc1gene restored theentire nuclease activity in the double deletion mutant, while thecomplementation of nuc2could only restore the original nuc2activity asthat of RNΔnuc1on toluidine blue-DNA agar. The deletion mutants andrespective complementation experiments illuminated the nuc2genefunction in expression thermonuclease in S. aureus and furtherdemonstrated the major role for nuc1in terms of thermonuclease activityin S. aureus.
4. A microarray method was used to analyze the transcriptionprofiles of S. aureus RN4220and its isogeneic△nuc1mutant and△nuc2mutant. The effected genes and pathways were compared and analyzed bythe contiguous genes and applied metabolites to the pathway. Microarrayanalysis revealed393genes expressed were significantly changed in thenuc1mutant in comparison to the parent strain. Meanwhile, only89geneswere significantly changed in the nuc2mutant, these genes were from similar pathway categories, such as amino acid metabolism, carbohydratemetabolism, replication and repair and nucleic acid metabolism andregulatory factors, however, only20of the changed genes were at thesame location. To validate of the microarray results,21differentiallyexpressed genes were selected and verified using quantitative real-timePCR. Particularly, the contiguous genes which were identified downregulated in the△nuc2mutant involved in the arginine-deiminase (ADI)pathway, while the ABC transporter related gene locus was up regulatedtogether. The purine, glycine, threonine, isoleucine metabolic pathwayswere also evolved when analyzed the contiguously regulated genes. In the△nuc1mutant, the altered genes evolved in the phosphotransferasesystem, lipid metabolism, and virulence regulatory factors were downregulated, and some of the urease genes and protease genes weresignificantly up-regulated. Generally, the△nuc1and△nuc2mutantsmade an impact in metabolic pathways, but the specific regulated geneswere different. The results implied that nuc1and nuc2may playrespectively roles to regulate various genes.
5. To provide a more complete analysis on the influence of twothermonuclease genes to the metabolism phenotype, Δnuc1and Δnuc2mutants were comprehensively tested using the phenotype microarray(PM) analysis of over1100phenotypes (including utilization of differentcarbon, nitrogen, phosphate, and sulfur sources; growth stimulation orinhibition by amino acids and other nutrients, osmolytes, pH andsusceptibility to antibiotics). Compared to the parental strain, the Δnuc2mutant weakened13phenotypes of1.5times or more, includingutilization of3kinds of carbon, sulfur sources,7kinds of nitrogensources,(dipeptides and ammonia metabolism). All altered phenotypes inthe Δnuc2mutant were defective, suggesting an important role of nuc2gene played in the metabolism procedure of Staphylococcus aureus. In contrast to the Δnuc2mutant,7out of9changed phenotypes werestimulated in the Δnuc1mutant, including utilization of nitrogen andphosphorus source, particularly enhanced growth in the acidic pH andamino acids supplies. The results of25antibiotic susceptibility testsshowed that the resistance to2-4cephalosporin antibiotics of Δnuc2mutant and Δnuc1mutant had increased, suggesting a relationship withthe expression differences of membrane proteins. Furthermore, thegrowth of Δnuc1mutant in pH5were obviously greater than that of theparental strain and the Δnuc2mutant in the growth curve, which may berelated with the increased biofilm formation of the Δnuc1mutant. Theenhanced metabolic phenotypes of the Δnuc1mutant suggest a bettersurvival in the biofilm formation than the wild type.
Taken together, the results of this study contribute to a new sight ofcharacterization, molecular function and metabolic networks of the twothermonuclease genes in S. aureus. It is helpful for a better understandingof regulation and the pathogenic mechanism of the thermonuclease genesin S. aureus.
1.采用生物信息学分析方法对金黄色葡萄球菌基因组中的nuc1、nuc2基因编码的蛋白质序列及结构进行比对，构建了不同种属间Nuc蛋白的进化树，揭示了金黄色葡萄球菌两个核酸酶在进化上的差异性。结果表明，Nuc2是一个在葡萄球菌属内进化较为保守的蛋白，而Nuc1则可能起源于其它菌属的水平转移。此外，在大肠杆菌中成功表达了两种核酸酶，对比研究了它们在体外表达时的酶活性特征，揭示了金黄色葡萄球菌Nuc2的酶学特征：最适反应温度为50℃，最适pH值为10，是一种非特异核糖核酸酶，适当浓度的Ca~(2+)、Mg~(2+)、DTT、β-mecaptoethanol、TritonX-100、Tween-20和Urea都能够提高Nuc2的酶活性，而Zn~(2+)、Mn~(2+)、EDTA和SDS则是Nuc2酶活性抑制剂。和Nuc1相比，体外表达的Nuc2酶活性较低，且对高温和重金属离子的耐受力明显低于Nuc1，这和Nuc2蛋白结构上不稳定的推测结果相符。本实验从序列、结构到酶活性特征上很好地区分了金黄色葡萄球菌两个重要的耐热核酸酶，对新近发现的耐热核酸酶Nuc2的特征作了较为详尽的研究，为其功能应用奠定了基础。
2.采用Taqman荧光定量PCR方法比较了两个耐热核酸酶基因在转录水平上的差异。通过对金黄色葡萄球菌不同生长阶段nuc1和nuc2基因mRNA表达水平的比较分析，发现nuc1和nuc2基因在生长过程中的表达模式并不相同：nuc1基因的表达在对数后期达到最大值，和耐热核酸酶活性测定结果一致；nuc2基因则在生长初期达到最大值，到对数后期反而降低。另外，通过测定金黄色葡萄球菌双组分调节子sae的缺失突变对nuc1和nuc2基因表达的影响，结果发现，sae基因缺失显著下调nuc1基因的表达；而nuc2基因在sae缺失突变株中表达几乎不受影响。金黄色葡萄球菌临床分离株中的nuc1基因表达变化差异明显，nuc2基因的表达则相对稳定。通过研究nuc1和nuc2基因在表达上的相关性，发现nuc1和nuc2基因受到不同的调控机制调控，且nuc1基因的表达是决定耐热核酸酶活性表型的主要因素。
3.利用微生物的同源重组双交换的遗传原理，成功构建了耐热核酸酶nuc2基因单缺失突变株和nuc1、nuc2基因双缺失突变株，并在分子水平进行了验证。突变株耐热核酸酶活性的测定结果表明，nuc2基因缺失后，酶活性比野生型降低，双突变株则检测不到耐热核酸酶活性。构建互补载体转化双突变株,互补菌株的酶活性测定结果发现，nuc1基因互补双缺失突变株能使其完全恢复到野生型的酶活水平，nuc2基因的互补只能恢复菌株nuc1基因突变后Nuc2的酶活表型。基因突变和互补实验确定了nuc2基因在金黄色葡萄球菌中具备耐热核酸酶活性表达功能，也进一步证实了nuc1基因在耐热核酸酶活性表型中占有主导地位的结论。
4.利用基因表达谱芯片技术研究了两种核酸酶基因突变后对金黄色葡萄球菌RN4220转录组的影响，采用连锁转录及基因代谢途径定位的新分析方法，对两种核酸酶基因突变所影响的差异基因进行分析，结果表明，nuc1基因缺失显著影响了393个基因转录水平的变化，而nuc2的缺失显著影响了89个基因转录水平的变化。虽然两组基因都涉及到菌株的氨基酸代谢、糖代谢、复制修复、核酸代谢及调控因子，但在相同位置发生差异变化的基因只有20个，且调控水平都不相同。挑选21个差异基因，采用荧光定量PCR验证了芯片结果的可靠性。值得注意的是，△nuc2缺失突变株和精氨酸脱亚胺酶途径相关的基因座均下调，和ABC转运子相关的基因座上调，连锁调控的代谢途径还涉及到嘌呤代谢、甘氨酸、苏氨酸、异亮氨酸等氨基酸代谢途径。△nuc1基因缺失主要下调了和磷酸转移酶系统、脂代谢及毒力调控因子的连锁基因的表达，上调了尿素酶基因及蛋白酶基因。△nuc1和△nuc2缺失突变株在转录水平分别调控了不同的基因表达，且各有分工。
5.为了更全面了解两种核酸酶基因的突变对于细胞代谢功能的影响，利用高通量的表型分析新技术--表型芯片(PM)研究了金黄色葡萄球菌两种耐热核酸酶突变株和亲本菌株RN4220的1100多种不同的表型，包括各种碳源、氮源、磷源、硫源的利用，对氨基酸及其他营养物质的代谢、渗透特征、pH及抗生素敏感性测试。和金葡亲本株相比，Δnuc2突变株的PM结果有13种表型减弱了1.5倍以上，包括对3种碳、硫源的利用，对7种氮源的利用，涉及氨基酸短肽及氨的代谢。突变株全部下调的表型说明了nuc2基因对金黄色葡萄球菌细胞的生理代谢的正常进行起着重要的作用； Δnuc1突变株的差异表型有9个，涉及氮源和磷源的代谢以及6个在酸性pH下的表型变化，和nuc2突变株表型不同的是，Δnuc1突变株中7种表型变化都是增强的，尤其是在酸性pH时突变株的生长和对氨基酸的利用上。25种抗生素敏感性测定结果发现，Δnuc2突变株和Δnuc1突变株对2-4种头孢类抗生素的抗性有所增加，这可能与膜蛋白的表达差异相关。生长曲线的测定结果表明，在酸性pH5条件下Δnuc1突变株的生长的明显高于Δnuc2菌株和野生型，且菌膜形成量明显增多，这与Δnuc1突变株在酸性条件下代谢表型的增强有重要的联系。
总之，本论文揭示了耐热核酸酶基因nuc2的特征及功能，对两个耐热核酸酶在金黄色葡萄球菌中表达调控机制进行了探索，为更好地了解金黄色葡萄球菌耐热核酸酶的作用机制具有重要意义。
Staphylococcus aureus is one of the most important pathogens thatcan cause food-borne disease, as well as nosocomial infections.Staphylococcal food poisoning and nosocomial infections are of majorconcerns in public health worldwide. S. aureus produces thermonucleasewhich is known as one of the most important virulence factors.Thermonuclease is widely used as a unique marker for detection andidentification of S. aureus. Our previous studies have revealed theexistence of two functional thermostable nucleases encoded by twodifferent genes (nuc1and nuc2) in the S. aureus genome. To characterizethe function of Nuc2enzyme, our research started from comparisonanalysis of Nuc2and Nuc1on the structure and feature. Furthermore, thefunctional and regulatory differences of the two thermonucleases in S.aureus were also charaterized. The main findings and results are listed asfollows:
1. Sequence alignments and phylogenetic analysis were carried outto study the evolution of Nuc1and Nuc2using bioinformatics tools. Theresults revealed that Nuc2protein was more conserved in thestaphylococci group compared with Nuc1protein, while the nuc1genemight have been acquired by lateral transfer from another pathogenicspecies. Two recombinant thermonucleases were successfully expressedin Escherichia coli, and their nuclease activities were further characterized and compared. The results showed that the optimal reactiontemperature and pH for recombinant Nuc2were50℃and pH10,respectively, and it was a sugar non-specific nucleases. The enzymaticactivity of Nuc2was stimulated in the presence of Ca~(2+), Mg~(2+),dithiothreitol, β-mecaptoethanol, TritonX-100, Tween-20, and urea;however, the activity decreased dramatically when exposed to heavymetals such as Zn~(2+)and Mn~(2+), and in the presence of EDTA or SDS.Nuc2showed a weaker activity, lower thermostability and differentsensitivity to the compounds compared with Nuc1, which showed acorrelation with the lack of a consensus pattern and changes of thehydrophobic core residues in the structure of Nuc2as predicted. Thisstudy determined important characteristics of the novel thermonuclease(Nuc2) in S. aureus and provided a discriminatory picture in therelationship on evolutionary and enzymatic differences of Nuc1and Nuc2by comparative analysis. This information helped us to better understandthe gene function and the potential application of this newthermonuclease gene.
2. To study the expression of these two genes, comparative mRNAanalysis of nuc1and nuc2was carried out by Taqman quantitative real-time PCR. Distinct expression patterns were observed at different growthstages. The maximum level of nuc1transcripts appeared at the post-exponential growth phase, and the transcription level correlated well withthermonuclease activity results. In contrast, nuc2transcripts were mostlyexpressed at the early exponential phase and declined at the post-exponential growth phase. Effects of sae mutation on the expression ofthermostable nuclease genes were also determined. The nuc1geneexpression was notably down-regulated in a sae mutant and the nuc2genewas barely changed in the sae mutant. Furthermore, unlike the expressionof nuc1that varied in three different S. aureus clinical strains, the transcription of nuc2remained relatively constant. Comparativeexpression analysis of two thermostable nuclease genes showed that nuc1and nuc2gene were not co-regulated in Staphylococcus aureus, and nuc1expression plays a more pronounced role in thermonuclease activitiesthan that of nuc2.
3. The nuc2knock out mutant and the nuc1and nuc2double deletionmutant of S. aureus were successfully constructed by homologousrecombination, and the mutants were confirmed on the molecular level.Thermonuclease assays revealed functional activity differences of nuc1and nuc2in S. aureus. Compared to the parental strain, RNΔnuc1andRNΔnuc2exhibited reduced activity zones on the plate. Thethermonuclease activity of the RNΔnuc2was higher than that of theRNΔnuc1on the toluidine blue-DNA agar. No activity was observed inthe nuc1and nuc2double deletion mutant. Complementation plasmidswere constructed and the complementation of the nuc1gene restored theentire nuclease activity in the double deletion mutant, while thecomplementation of nuc2could only restore the original nuc2activity asthat of RNΔnuc1on toluidine blue-DNA agar. The deletion mutants andrespective complementation experiments illuminated the nuc2genefunction in expression thermonuclease in S. aureus and furtherdemonstrated the major role for nuc1in terms of thermonuclease activityin S. aureus.
4. A microarray method was used to analyze the transcriptionprofiles of S. aureus RN4220and its isogeneic△nuc1mutant and△nuc2mutant. The effected genes and pathways were compared and analyzed bythe contiguous genes and applied metabolites to the pathway. Microarrayanalysis revealed393genes expressed were significantly changed in thenuc1mutant in comparison to the parent strain. Meanwhile, only89geneswere significantly changed in the nuc2mutant, these genes were from similar pathway categories, such as amino acid metabolism, carbohydratemetabolism, replication and repair and nucleic acid metabolism andregulatory factors, however, only20of the changed genes were at thesame location. To validate of the microarray results,21differentiallyexpressed genes were selected and verified using quantitative real-timePCR. Particularly, the contiguous genes which were identified downregulated in the△nuc2mutant involved in the arginine-deiminase (ADI)pathway, while the ABC transporter related gene locus was up regulatedtogether. The purine, glycine, threonine, isoleucine metabolic pathwayswere also evolved when analyzed the contiguously regulated genes. In the△nuc1mutant, the altered genes evolved in the phosphotransferasesystem, lipid metabolism, and virulence regulatory factors were downregulated, and some of the urease genes and protease genes weresignificantly up-regulated. Generally, the△nuc1and△nuc2mutantsmade an impact in metabolic pathways, but the specific regulated geneswere different. The results implied that nuc1and nuc2may playrespectively roles to regulate various genes.
5. To provide a more complete analysis on the influence of twothermonuclease genes to the metabolism phenotype, Δnuc1and Δnuc2mutants were comprehensively tested using the phenotype microarray(PM) analysis of over1100phenotypes (including utilization of differentcarbon, nitrogen, phosphate, and sulfur sources; growth stimulation orinhibition by amino acids and other nutrients, osmolytes, pH andsusceptibility to antibiotics). Compared to the parental strain, the Δnuc2mutant weakened13phenotypes of1.5times or more, includingutilization of3kinds of carbon, sulfur sources,7kinds of nitrogensources,(dipeptides and ammonia metabolism). All altered phenotypes inthe Δnuc2mutant were defective, suggesting an important role of nuc2gene played in the metabolism procedure of Staphylococcus aureus. In contrast to the Δnuc2mutant,7out of9changed phenotypes werestimulated in the Δnuc1mutant, including utilization of nitrogen andphosphorus source, particularly enhanced growth in the acidic pH andamino acids supplies. The results of25antibiotic susceptibility testsshowed that the resistance to2-4cephalosporin antibiotics of Δnuc2mutant and Δnuc1mutant had increased, suggesting a relationship withthe expression differences of membrane proteins. Furthermore, thegrowth of Δnuc1mutant in pH5were obviously greater than that of theparental strain and the Δnuc2mutant in the growth curve, which may berelated with the increased biofilm formation of the Δnuc1mutant. Theenhanced metabolic phenotypes of the Δnuc1mutant suggest a bettersurvival in the biofilm formation than the wild type.
Taken together, the results of this study contribute to a new sight ofcharacterization, molecular function and metabolic networks of the twothermonuclease genes in S. aureus. It is helpful for a better understandingof regulation and the pathogenic mechanism of the thermonuclease genesin S. aureus.
引文
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