The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation,membrane binding,lipid-specific domains and channel activities

SecA is an essential multifunctional protein for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane, the detailed mechanism for this process remains unclear. In this study we constructed a series of SecA N-terminal deletions and identified two specific domains crucial for initial SecA/membrane interactions. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of a SecA ts mutant. However, deletions of N-terminal aminoacyl residues 23–25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity. All together, the results indicate that the N-terminal aminoacyl residues 23–25 play a critical role for SecA binding to membranes and that the N-terminal limit of SecA for activity is at the 25th amino acid.     

SecAAA trimer is fully functional as SecAA dimer in the membrane: Existence of higher oligomers?

SecA is an essential ATPase in bacterial Sec-dependent protein translocation pathway, and equilibrates between monomers and dimers in solution. The question of whether SecA functions as monomers or dimers in membranes during the protein translocation is controversial. We previously constructed a tail-to-head SecAA tandem dimer, and showed it is fully functional by complementation in vivo and protein translocation in vitro, indicating that SecA can function at least as a dimer in the membrane without dissociating into monomers. In this study, we further constructed genetically a tail-to-head SecAAA trimer, which is functional in complementing a temperature-sensitive secA mutant. The purified SecAAA trimer per protomer is fully active as SecAA tandem dimers in ATPase activity, in protein translocation in vitro and in ion channel activities in the oocytes. With these functional tail-to-head trimer SecAAA and tandem SecAA, we examined their surface topology in the presence of liposomes using AFM. As expected, the soluble SecAAA without lipids are larger than SecAA. However, the ring/pore structures of SecAAA trimers were, surprisingly, almost identical to the SecA 2-monomers and SecAA dimers, raising the intriguing possibility that the SecA may exist and function as hexamer ring-structures in membranes. Cross-linking with formaldehyde showed that SecA, SecAA and SecAAA could form larger oligomers, including the hexamers. The molecular modeling simulation shows that both tail-to-head and tail-to-tail hexamers in the membranes are possible.     

Lipid productivity and fatty acid composition-guided selection of Chlorella strains isolated from Malaysia for biodiesel production

The need to develop biomass-based domestic production of high-energy liquid fuels (biodiesel) for transportation can potentially be addressed by exploring microalgae with high lipid content. Selecting the strains with adequate oil yield and quality is of fundamental importance for a cost-efficient biofuel feedstock production based on microalgae. This work evaluated 29 strains of Chlorella isolated from Malaysia as feedstock for biodiesel based on volumetric lipid productivity and fatty acid profiles. Phylogenetic studies based on 18S rRNA gene revealed that majority of the strains belong to true Chlorella followed by Parachlorella. The strains were similarly separated into two groups based on fatty acid composition. Of the 18 true Chlorella strains, Chlorella UMACC187 had the highest palmitic acid (C16:0) content (71.3?±?4.2 % total fatty acids, TFA) followed by UMACC84 (70.1?±?0.7 %TFA), UMACC283 (63.8?±?0.7 %TFA), and UMACC001 (60.3?±?4.0 %TFA). Lipid productivity of the strains at exponential phase ranged from 34.53 to 230.38 mg L^?1 day^?1, with Chlorella UMACC050 attaining the highest lipid productivity. This study demonstrated that Chlorella UMACC050 is a promising candidate for biodiesel feedstock production.     

Structural Characterization of the Complex of SecB and Metallothionein-Labeled proOmpA by Cryo-Electron Microscopy

ProOmpA is a preprotein that is translocated across the plasma membrane by the general secretory pathway in Escherichia coli. The molecular chaperon SecB in Sec pathway can recognize and bind proOmpA for its translocation. However, the structure of the SecB/proOmpA complex remains unknown. Here, we constructed an uncleavable proOmpA fused with metallothionein at its C-terminus and labeled it with metals in vitro for the study of cryo-electron microscopy. Using single particle cryo-electron microscopy, we reconstructed 3D structure of the stable SecB/proOmpA complex. The structure shows that the major portion of preprotein locates on one side of SecB tetramer, resulting in an asymmetric binding pattern. This work also provides a possible approach to the structure determination of small protein complexes by cryo-electron microscopy.     

Discovery of the first SecA inhibitors using structure-based virtual screening

Bacterial protein secretion is a critical and complex process. The Sec machinery provides a major pathway for protein translocation across and integration into the cellular membrane in bacteria. Small molecule probes that perturb the functions of individual member proteins within the Sec machinery will be very important research tools as well as leads for future antimicrobial agent development. Herein we describe the discovery of inhibitors, through virtual screening, that specifically act on SecA ATPase, which is a critical member of the Sec system. These are the very first inhibitors reported for intrinsic SecA ATPase.     

Full-length Escherichia coli SecA dimerizes in a closed conformation in solution as determined by cryo-electron microscopy

SecA is an obligatory component of the Escherichia coli general secretion pathway. However, the oligomeric structure of SecA and SecA conformational changes during translocation processes are still unclear. Here we obtained the three-dimensional structure of E. coli wild-type full-length SecA in solution by single particle cryo-electron microscopy and determined its oligomeric organization. In this structure, SecA occurs as a dimer in which the two protomers are arranged in an antiparallel mode, with a novel electrostatic interface, and both protomers are in closed conformation. The system developed here may provide a promising technique for studying dynamic structural changes in SecA.     

Additional in vitro and in vivo evidence for SecA functioning as dimers in the membrane: dissociation into monomers is not essential for protein translocation in Escherichia coli

SecA is an essential component in the Sec-dependent protein translocation pathway and, together with ATP, provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. Previous studies established that SecA undergoes monomer-dimer equilibrium in solution. However, the oligomeric state of functional SecA during the protein translocation process is controversial. In this study, we provide additional evidence that SecA functions as a dimer in the membrane by (i) demonstration of the capability of the presumably monomeric SecA derivative to be cross-linked as dimers in vitro and in vivo, (ii) complementation of the growth of a secA(Ts) mutant with another nonfunctional SecA or (iii) in vivo complementation and in vitro function of a genetically tandem SecA dimer that does not dissociate into monomers, and (iv) formation of similar ring-like structures by the tandem SecA dimer and SecA in the presence of lipid bilayers. We conclude that SecA functions as a dimer in the membrane and dissociation into monomers is not necessary during protein translocation.     

Molecular differentiation of two morphological variants of Gracilaria salicornia

Xia in 1986 combined Gracilaria salicornia, G. canaliculata (G. crassa), G. cacalia and G. minor into one species: G.salicornia. Two morphological variants of G. salicornia were collectedfrom different localities in Malaysia. Variant A collected from Morib,Selangor grew on the roots of Avicennia. The samples showed absenceof main axis; segmented constrictions throughout; cylindrical or slightlycompressed thalli. Variant B was collected from the mudflats of TanjungTuan, growing on rocks, coral or forming mats on the mud. Plants showedabsence of main axis; segments were not constricted throughout the plant(if present only slightly articulated at the upper part), branching wasdichotomous or irregular; cylindrical or slightly compressed thalli. Thetechnique of Random Amplified Polymorphic DNA analysis (RAPD) wasused to investigate molecular characteristics of the two variants. Out ofsixty Operon primers that were screened, four primers, OPA 1, OPA 10,OPA 11 and OPK 7 were able to give polymorphism. The fingerprintsgenerated were stable and reproducible on repeated analysis. The DNAfingerprints generated were visually analysed and clustering analysis wascarried out using GelCompar 4.0. The matrix of similarities was based onthe Dice coefficients (S_D) and the cluster analysis was carried outusing the unweighted pair group method using arithmetic averages(UPGMA). DNA analysis showed that two primers (OPA 01, CAGGCCCTTC and OPK 07, AGCGAGCAAG) were able to differentiate the two variants.     

Effect of four chlorophytes on larval survival, development and adult body size of the mosquito Aedes aegypti

The effect of four microalgal chlorophytes, Chlorella vulgaris,Scenedesmus quadricauda, Chlorococcum sp. and Ankistrodesmus convolutus, on the survival, larval development and adultbody size of the mosquito Aedes aegypti was investigated. The percentage mortality of larvae fed with C. vulgaris, Chlorococcum sp.or S. quadricauda after six days was 100%, 84% and 88%,respectively. Delayed pupation and body size reduction of the mosquitoesfed with C. vulgaris, Chlorococcum sp. and S. quadricaudawere observed. In contrast, larvae fed with A. convolutus werebigger than those fed with normal insectory feed. The study showed thatC. vulgaris, S. quadricauda and Chlorococcum sp. have potentialto be used as larvicidal agents.