Hottmann Isabel, Mayer Valentina M T, Tomek Markus B, Friedrich Valentin, Calvert Matthew B, Titz Alexander, Schäffer Christina, Mayer Christoph
Microbiology and Biotechnology, Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Department of Biology, Eberhard Karls Universität Tübingen, Tübingen, Germany.
NanoGlycobiology Unit, Department of NanoBiotechnology, Universität für Bodenkultur Wien, Vienna, Austria.
Front Microbiol. 2018 Jan 26;9:19. doi: 10.3389/fmicb.2018.00019. eCollection 2018.
is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for In between the respective genes on the genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 10-fold higher catalytic efficiency (/ ) for MurNAc than for acetylglucosamine (GlcNAc) with values of 10.5 s and 0.1 s and values of 200 μM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition ( = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of , a kinase deletion mutant () was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 μg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 μg/ml MurNAc for optimal growth. These data reveal that readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.
是一种厌氧的革兰氏阴性口腔病原体，在与牙周炎相关的多物种牙龈生物膜中大量繁殖。该细菌对通常必不可少的细菌细胞壁糖乙酰胞壁酸（MurNAc）营养缺陷，因此，其生长和细胞形态维持严格依赖于外源性MurNAc供应。最近报道了一种MurNAc转运蛋白（Tf_MurT；Tanf_08375）和将MurNAc-6-磷酸转化为GlcNAc-6-磷酸的醚酶MurQ的直系同源物（Tf_MurQ；Tanf_08385）。在基因组上的各个基因之间，定位了一个假定的激酶基因。在本研究中，假定的激酶（Tf_MurK；Tanf_08380）作为重组蛋白产生并进行了生化表征。动力学研究表明，Tf_MurK是一种6-激酶，对MurNAc具有严格的底物特异性，对MurNAc的催化效率（/）比对乙酰葡糖胺（GlcNAc）高6×10倍，其值分别为10.5 s和0.1 s，值分别为200μM和116 mM。酶动力学数据表明，Tf_MurK受到底物抑制（=4.2 mM）。为了评估Tf_MurK在细胞壁代谢中的作用，构建了一个激酶缺失突变体（）。该突变体在指数生长期在细胞内积累MurNAc，表明其具有摄取MurNAc的能力，但无法分解代谢MurNAc。在稳定期，突变体中的MurNAc水平降低，而肽聚糖前体UDP-MurNAc-五肽的水平高度升高。此外，根据扫描电子显微镜证据，突变体在从健康的杆状细胞转变为梭形细胞之前，对培养基中低MurNAc浓度（低于0.5μg/ml）的耐受性更强，而亲本菌株需要>1μg/ml MurNAc才能实现最佳生长。这些数据表明，能够轻易分解代谢外源性MurNAc，但同时将一部分糖引导至肽聚糖生物合成中。缺失会阻断MurNAc分解代谢，并使MurNAc仅用于肽聚糖生物合成，从而在MurNAc耗尽的培养基中产生生长优势。这项工作增进了我们对细胞壁代谢的理解，并可能为牙周炎治疗中的先导化合物发现开辟新途径。
