Characterization of cytochrome mutants for pellicle formation in Shewanella onedensis MR-1 LIANG Yi-li(梁伊丽) 1, 2 , HE Zhi-li(贺志理) 2 , GAO Hai-chun(高海春) 2 , QIU Guan-zhou(邱冠周) 1 , ZHOU Ji-zhong(周集中) 1, 2 , LIU Xue-duan(刘学端) 1 1. School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China; 2. Institute for Environmental Genomics and Department of Botany and Microbiology, University of Oklahoma, Norman 73019, USA Received 9 March 2009; accepted 9 April 2009 Abstract: Biofilm systems are effective for biosorption of metal ions. Shewanella oneidensis MR-1, a Gram-negative facultative anaerobe, is a natural pellicle-like biofilm former. The mechanisms of pellicle formation by S. oneidensis MR-1 have not yet been understood. 17 S. oneidensis MR-1 deletion mutants, including 12 c-type cytochromes were generated and tested if they were involved in pellicle formation. The results show that ∆SO4666, ∆SO1777, ∆SO1782, ∆SO2361 and ∆SO2363 have varying deficiency in pellicle formation. The deletion mutant ∆SO4666 cannot form a pellicle under non-shake conditions, suggesting that it may play an important role in pellicle formation by S. oneidensis MR-1. Overall, these data suggest a very complex picture of aerobic respiration by S. oneidensis MR-1. Key words: cytochrome; mutant; electron transport; pellicle; biofilms 1 Introduction Considerable studies have been focused in recent years upon the field of biosorption for the removal of metal ions from aqueous effluents[1]. Biofilms promise to be suitable systems for the treatment of metal ions since microorganisms that absorb metals stabilize the extracellular polymeric matrix by the combined action of chemical, physical and physiological phenomena that are, in some instances, linked to phenotypic variation among the constituent biofilm cells[2]. Shewanella oneidensis MR-1, a facultative Gram-negative anaerobe with a remarkable respiratory versatility, is a natural biofilm former. In recent years, this organism has attracted a great deal of interest due to its potential applications in bioremediation of metal contaminants in the environment. S. oneidensis MR-1 has been extensively studied, including the development of the canonical biofilms [3−9]. S. oneidensis MR-1 possesses a complex electron- transport system. Cytochromes, heme-containing proteins are the main components of the respiratory electron transport chains[10]. Approximately, 42 cytochrome c genes are annotated based on sequence analysis and 41 are likely to be functional in comparison with five to seven in E. coli and other enteric bacteria [11], and most of them are more or less mobile electron transfer proteins on the periplasmic side of the membrane[12]. A fraction of the Shewanella cytochrome genes have been characterized at the molecular level under anaerobic condition[13]. Since c-type cytochromes are essential for energy metabolism, their mutation will also directly affect the aerobic electron transport network. However, the role of most cytochromes under aerobic conditions remains unknown. In this work, the mechanisms of pellicle formation in S. oneidensis MR-1 are explored. We generated and characterized 12 cytochrome mutants and 5 other mutants related to protein secretion systems and regulatory proteins, and found that some cytochromes show deficiency in pellicle formation under aerobic conditions. These data represent the first identification of genes specifically related to pellicle formation and an Foundation item: Project(50321402) supported by Chinese Science Foundation for Distinguished Group; Project(30428014) supported by National Science Fund for Distinguished Young Scholars in Hong Kong and Abroad Corresponding author: ZHOU Ji-zhong; E-mail: jzhou@ou.edu; LIU Xue-duan; Tel: +86-731-8830546; E-mail: xueduanliu@yahoo.com DOI: 10.1016/S1003-6326(08)60336-2