Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Università di Palermo Viale delle Scienze, ed, 16, Parco d'Orleans II, Palermo, 90128, Italy.
Microb Cell Fact. 2012 Nov 23;11:152. doi: 10.1186/1475-2859-11-152.
A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics. In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having a high rhamnose content and metal-binding properties, whose biotechnological applications were proven as very relevant.
Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun, combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from these differential studies peculiar levels of outer membrane proteins, key regulatory factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar biosynthesis or required for citrate fermentation and stress response during anaerobic growth on Fe(III)-citrate were revealed. The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production.
Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.
先前从黄铁矿矿排水中分离并命名为 BAS-10 的细菌菌株被初步鉴定为产酸克雷伯氏菌。与其他肠杆菌不同，BAS-10 能够以 Fe(III)-柠檬酸盐为唯一碳源和能源生长，产生乙酸和 CO2，同时将 Fe(III)还原为 Fe(II)，并表现出异常的生理特性。事实上，在这种生长条件下，BAS-10 会产生一种具有高鼠李糖含量和金属结合特性的胞外多糖 (EPS)，其生物技术应用已被证明非常相关。
基于 16S rDNA 序列的进一步系统发育分析，明确证实 BAS-10 属于产酸克雷伯氏菌属。为了使这种不寻常肠杆菌的生化特性合理化，我们结合了 2D-DIGE 分析和质谱程序来研究其蛋白质组变化：i）在有氧或无氧条件下以 Fe(III)-柠檬酸盐为唯一碳源培养；ii）在无氧条件下以 Na(I)-柠檬酸盐或 Fe(III)-柠檬酸盐为唯一碳源培养。结合这些差异研究的数据，揭示了外膜蛋白、碳氮代谢关键调控因子、三羧酸循环和糖生物合成相关酶以及柠檬酸发酵和厌氧条件下 Fe(III)-柠檬酸生长时的应激反应所需酶的特殊水平。蛋白质的差异调节似乎确保了有效的细胞生长，同时通过适应代谢和生化过程来生产 EPS，从而应对铁毒性并优化能量产生。
差异蛋白质组学为生物工程上有前途的肠杆菌利用 Fe(III)-柠檬酸盐提供了必要的分子机制见解，同时还揭示了可以针对高产 EPS 生产菌株的理性设计进行靶向的基因。
