Robin Ristl, Bettina Janesch, Julia Anzengruber, Agnes Forsthuber, Johanna Blaha, Paul Messner, Christina Schäffer
Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacterium Paenibacillus alvei CCM 2051T is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the P. alvei S-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosine O-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present in P. alvei CCM 2051T, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic of O-oligosaccharyl:protein transferases (O-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy_C motif resulted in loss of WsfB function evidenced in reconstitution experiments in P. alvei ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in Escherichia coli CWG702 and Salmonella enterica SL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterial O-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase of P. alvei, and a promising candidate for the first O-OTase reported in Gram-positives.
表面(S)层蛋白质是研究细菌中蛋白质糖基化的模型系统，同时由于其二维自组装能力，为纳米生物技术设计新颖的糖功能化模块提供了希望。了解革兰氏阳性细菌芽孢杆菌CCM 2051T中s层聚糖生物合成的机制对于其s层的糖功能化是必要的。本文对来自肺泡藻s层糖基化基因位点的推定寡糖s层蛋白转移酶WsfB进行了表征。该酶被提议催化糖基化途径的最后一步，将拉长的s层聚糖转移到不同的酪氨酸o糖基化位点。研究表明，WsfB基因敲除可以消除P. alvei CCM 2051T中s层蛋白SpaA的糖基化，但不能消除其他糖蛋白的糖基化，从而将其作用局限于s层糖基化途径。从绿色荧光蛋白和磷酸酶A融合到WsfB的定义截断的活性测量中推断出781个氨基酸WsfB蛋白的跨膜拓扑模型。该模型显示了13个膜跨越螺旋，它们具有o -寡糖的Wzy_C结构域特征:位于中央胞质外环的蛋白质转移酶(O-OTases)，这两者都与革兰氏阴性菌的OTases的拓扑结构相比较。Wzy_C基序的突变导致WsfB功能的丧失，这在肺泡p.a vei ΔWsfB细胞的重建实验中得到了证实。在大肠杆菌CWG702和肠沙门氏菌SL3749中，利用WsfB将异源寡糖转移到其天然s层靶蛋白上的尝试没有成功，这可能是由于WsfB的严格功能导致的，这些蛋白应该在一定程度上模仿天然宿主的脂联寡糖底物。综上所述，WsfB具有细菌O-OTase的所有特征，使其成为P. alvei低聚糖s层蛋白转移酶的最有可能的候选者，并且是革兰氏阳性报告的第一个O-OTase的有希望的候选者。
{"title":"Description of a Putative Oligosaccharyl:S-Layer Protein Transferase from the Tyrosine <i>O</i>-Glycosylation System of <i>Paenibacillus alvei</i> CCM 2051<sup>T</sup>.","authors":"Robin Ristl, Bettina Janesch, Julia Anzengruber, Agnes Forsthuber, Johanna Blaha, Paul Messner, Christina Schäffer","doi":"10.4236/aim.2012.24069","DOIUrl":"https://doi.org/10.4236/aim.2012.24069","url":null,"abstract":"<p><p>Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacterium <i>Paenibacillus alvei</i> CCM 2051<sup>T</sup> is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the <i>P. alvei</i> S-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosine <i>O</i>-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present in <i>P. alvei</i> CCM 2051<sup>T</sup>, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic of <i>O</i>-oligosaccharyl:protein transferases (<i>O</i>-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy_C motif resulted in loss of WsfB function evidenced in reconstitution experiments in <i>P. alvei</i> ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in <i>Escherichia coli</i> CWG702 and <i>Salmonella enterica</i> SL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterial <i>O</i>-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase of <i>P. alvei</i>, and a promising candidate for the first <i>O</i>-OTase reported in Gram-positives.</p>","PeriodicalId":7355,"journal":{"name":"Advances in Microbiology","volume":"2 4","pages":"537-546"},"PeriodicalIF":0.0,"publicationDate":"2012-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397953/pdf/emss-62937.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33115048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Polymyxins are often considered as a last resort to treat multidrug resistant P. aeruginosa but polymyxin resistance has been increasingly reported worldwide in clinical isolates. Polymyxin resistance in P. aeruginosa is known to be associated with alterations in either PhoQ or PmrB. In this study, mutant strains of P. aeruginosa carrying amino acid substitution, a single and/or dual inactivation of PhoQ and PmrB were constructed to further understand the roles of PhoQ and PmrB in polymyxin susceptibility. Polymyxin B resistance was caused by both inactivation and/or amino acid substitutions in PhoQ but by only amino acid substitutions of PmrB. Alterations of both PhoQ and PmrB resulted in higher levels of polymyxin B resistance than alteration of either PhoQ or PmrB alone. These results were confirmed by time-killing assays suggesting that high-level polymyxin resistance in P. aeruginosa is caused by alterations of both PhoQ and PmrB.
{"title":"Differential Role of Two-Component Regulatory Systems (<i>phoPQ</i> and <i>pmrAB</i>) in Polymyxin B Susceptibility of <i>Pseudomonas aeruginosa.</i>","authors":"Daniel Owusu-Anim, Dong H Kwon","doi":"10.4236/aim.2012.21005","DOIUrl":"https://doi.org/10.4236/aim.2012.21005","url":null,"abstract":"<p><p>Polymyxins are often considered as a last resort to treat multidrug resistant <i>P. aeruginosa</i> but polymyxin resistance has been increasingly reported worldwide in clinical isolates. Polymyxin resistance in <i>P. aeruginosa</i> is known to be associated with alterations in either PhoQ or PmrB. In this study, mutant strains of <i>P. aeruginosa</i> carrying amino acid substitution, a single and/or dual inactivation of PhoQ and PmrB were constructed to further understand the roles of PhoQ and PmrB in polymyxin susceptibility. Polymyxin B resistance was caused by both inactivation and/or amino acid substitutions in PhoQ but by only amino acid substitutions of PmrB. Alterations of both PhoQ and PmrB resulted in higher levels of polymyxin B resistance than alteration of either PhoQ or PmrB alone. These results were confirmed by time-killing assays suggesting that high-level polymyxin resistance in <i>P. aeruginosa</i> is caused by alterations of both PhoQ and PmrB.</p>","PeriodicalId":7355,"journal":{"name":"Advances in Microbiology","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2012-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859615/pdf/nihms528316.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31965972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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