Identification And Functional Studies Of Outer Membrane Proteins Of Anabaena Sp. PCC 7120

Anabaena sp. strain PCC7120 is a filamentous cyanobacterium in which, in response to deprivation of fixed nitrogen, vegetative cells at semiregular intervals differentiate into nitrogen-fixing cells called heterocysts. For the investigation of cell differentiation and pattern formation in a one-dimension organism, Anabaena has been used as the model species. Outer membrane of Anabaena and other cyanobacteria is the first barrier for nutrition uptake, and in Anabaena even affects the development of heterocyst envelope. But until now, few functional studies have been done about outer membrane proteins of cyanobacteria. In this study, outer membrane of Anabaena sp. PCC7120 was isolated, and some outer membrane proteins with high expression level or involved in iron uptake were identified using 2-DE and mass spectrometry. Reporter gene of bacterial luciferase (luxAB) was used to analyze transcription of genes encoding the outer membrane proteins. Four genes were inactivated by targeted disruption, among which two genes were found involved in Ca2+ uptake.The experimentation and results are described in 3 parts:(1) Purification of the outer membrane and identification of outer membrane proteins from Anabaena sp. PCC7120. The outer membrane from Anabaena 7120 was isolated using aqueous polymer two-phase partitioning in combination with sucrose density gradient ultra-centrifugation with very few cross-contaminations by plasma and thylakoid membranes. The purity of the outer membrane was verified by absorption spectra and western-blot using antibodies against membrane-specific marker proteins. Eight proteins were identified using 2-DE and MOLDI-TOF MS or ESI-Q-TOF MS.(2) The study of outer membrane proteins involved in iron uptake. 2-DE was used to analyze outer membrane proteins from cultures grown under iron-sufficient and iron-deficient conditions, and the results showed that five of eight outer membrane proteins involved in iron uptake were up-regulated by iron deprivation, while the other three having no obvious change. By studying the expression of the eight genes using luxAB, it was demonstrated that transcriptional complementarity relationship existed among the eight genes.(3) The study of outer membrane proteins involved in calcium uptake. Mutants were obtained by gene disruption of four genes encoding outer membrane proteins with high expression level. The preliminary screening results indicated that in NO3--deficient media, mutant all3983::C.CE2 grew at a slightly lower rate than the wild type. The expression of all3983 was up-regulated in another mutant alr2269::C.CE2. By using luxAB, it was found that the expression of all3983 in mutant alr2269::C.CE2 was gradually up-regulated with the nutrient consumption. Removal of individual nutrient element from BG-11 showed that the regulation of all3983 was mainly dependent on the Ca2+ concentration. Compared to the wild-type, mutant alr2269::C.CE2 grew slowly in BG-11 with 1/50 Ca2+, and mutant all3983::C.CE2 grew slowly in BG-11 with 1/1000 Ca2+. All these showed that outer membrane proteins of Alr2269 and All3983 probably played an important role in Ca2+ uptake.The main conclusions of this study are as follows:(1) Outer membrane from the Anabaena PCC 7120 could be isolated efficiently by aqueous polymer two-phase partitioning in combination with sucrose density gradient ultra-centrifugation.(2) Five of eight outer membrane proteins involved in iron uptake were up-regulated by iron deprivation, and in transcriptional level the regulation of these genes appeared to be complementary with each other.(3) Alr2269 and All3983 were probably involved in Ca2+ uptake.