Goethe Elke, Gieseke Ayla, Laarmann Kristin, Lührs Janita, Goethe Ralph
Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany
Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany.
J Bacteriol. 2021 May 1;203(9). doi: 10.1128/JB.00049-21. Epub 2021 Mar 15.
Zinc uptake in bacteria is essential to maintain cellular homeostasis and survival. ZnuABC is an important zinc importer of numerous bacterial genera, which is expressed to restore zinc homeostasis when the cytosolic concentration decreases beyond a critical threshold. Upon zinc limitation the fast-growing nonpathogenic organism (MSMEG) as well as the ruminant pathogen subsp. (MAP) increases expression of genes encoding ZnuABC homologues, but also of genes encoding other transporters. This suggests an involvement of these transporters in zinc homeostasis. Here we characterized the putative zinc transporters of MSMEG (ZnuABC and ZnuABC2) and MAP (ZnuABC, MptABC, and MAP3774-76). Deletion of either ZnuABC or ZnuABC2 in MSMEG did not lead to growth defects, but to an increased expression of zinc marker genes in MSMEGΔABC, indicating cytosolic zinc limitation. However, chromatin immunoprecipitation proved direct binding of the global zinc regulator Zur to promoter regions of both ABC and ABC2. Simultaneous deletion of both transporters caused severe growth defects, which could be restored either by homologous complementation with single ZnuABC transporters or supplementation of growth media with zinc but not iron, manganese, cobalt, or magnesium. Heterologous complementation of the double mutant with MAP transporters also resulted in reconstitution of growth. Nonradioactive FluoZin-3AM zinc uptake assays directly revealed the competence of all transporters to import zinc. Finally, structural and phylogenetic analyses provided evidence of a novel class of ZnuABC transporters represented by the ZnuABC2 of MSMEG, which is present only in actinobacteria, mainly in the genera , and fast growing Zinc is necessary for bacterial growth but simultaneously toxic when in excess. Hence, bacterial cells have developed systems to alter intracellular concentration. Regulation of these systems is primarily executed at transcriptional level by regulator proteins which sense femtomolar changes in the zinc level. In environmental and pathogenic mycobacteria zinc starvation induces expression of common zinc import systems such as the ZnuABC transporter, but also of other additional not yet characterized transport systems. In this study, we characterized the role of such systems in zinc transport. We showed that transport systems of both species whose transcription is induced upon zinc starvation can exchangeably restore cellular zinc homeostasis in transporter deficient mutants by transporting zinc into the cell.
细菌摄取锌对于维持细胞内稳态和生存至关重要。ZnuABC是众多细菌属中一种重要的锌导入蛋白，当胞质浓度降至临界阈值以下时，它会表达以恢复锌稳态。在锌限制条件下，快速生长的非致病生物（耻垢分枝杆菌）以及反刍动物病原体亚种（副结核分枝杆菌）会增加编码ZnuABC同源物的基因以及编码其他转运蛋白的基因的表达。这表明这些转运蛋白参与了锌稳态的维持。在此，我们对耻垢分枝杆菌（ZnuABC和ZnuABC2）和副结核分枝杆菌（ZnuABC、MptABC和MAP3774 - 76）的假定锌转运蛋白进行了表征。在耻垢分枝杆菌中删除ZnuABC或ZnuABC2均未导致生长缺陷，但在耻垢分枝杆菌ΔABC中锌标记基因的表达增加，表明胞质锌受限。然而，染色质免疫沉淀证明全局锌调节因子Zur直接结合到ABC和ABC2的启动子区域。同时删除这两种转运蛋白会导致严重的生长缺陷，这可以通过用单个ZnuABC转运蛋白进行同源互补或在生长培养基中补充锌而非铁、锰、钴或镁来恢复。用副结核分枝杆菌转运蛋白对双突变体进行异源互补也导致了生长的恢复。非放射性FluoZin - 3AM锌摄取测定直接揭示了所有转运蛋白摄取锌的能力。最后，结构和系统发育分析提供了一类以耻垢分枝杆菌的ZnuABC2为代表的新型ZnuABC转运蛋白的证据，该蛋白仅存在于放线菌中，主要存在于分枝杆菌属、诺卡氏菌属和快速生长的棒状杆菌属中。锌对于细菌生长是必需的，但过量时同时具有毒性。因此，细菌细胞已发展出改变细胞内浓度的系统。这些系统的调节主要在转录水平由感知锌水平飞摩尔变化的调节蛋白执行。在环境和致病性分枝杆菌中，锌饥饿会诱导常见锌导入系统如ZnuABC转运蛋白以及其他尚未表征的转运系统的表达。在本研究中，我们表征了此类系统在锌转运中的作用。我们表明，在锌饥饿时其转录被诱导的两种物种的转运系统可以通过将锌转运到细胞中，可互换地恢复转运蛋白缺陷突变体中的细胞锌稳态。
