The in vivo and in vitro Reconstitution of Pigment-protein Complexes,and its Implication in Acquiring a New System

Reconstitution is one of the most fundamental and powerful tools to investigate pigment—protein complexes, for example, light-harvesting complexes and reaction center complexes. Two reconstitution methods, in vitro and in vivo, have been applied to complexes. In vitro reconstitution methods were first developed using isolated proteins and pigments, and recently using over-expressed proteins. This method enables analysis of pigment binding, pigment stoichiometry, and protein flexibility when accepting extrinsic pigments, however, it has not yet been successfully applied to the core antenna system. In vivo reconstitution, which was developed using genetic modifications, is applicable even on core systems. In this Review, the in vivo reconstitution is mainly considered on the basis of the in vitro reconstitution, because the former was a recent development and will be expanded to many systems. When genes for a new pigment are acquired and expressed, the new pigment is incorporated into a pre-exiting complex(es) and becomes functional when it is accepted by this complex(es), or abandoned if it is not. This process is postulated to occur during the evolutionary process(es) of antenna and reaction centers, and it is now possible to reproduce this evolutionary developmental pathway(s). Several examples of in vivo reconstitution are given and considered from the viewpoint of evolutionary implication with regards to the antenna and reaction centers.This revised version was published online in October 2005 with corrections to the Cover Date.     

Shigella invasion of macrophage requires the insertion of IpaC into the host plasma membrane. Functional analysis of IpaC.

Shigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death. The bacterial strategy of macrophage infection, however, remains largely speculative. Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria. When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000. J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester. Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake of Escherichia coli. The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining. The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117-169, which contains a putative membrane-spanning sequence. Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial.     

Light-Harvesting for Photosynthesis in Four Strains of the Red Alga Porphyra yezoensis Having Different Phycobilin Contents

The light-harvesting system of photosynthesis was studied infour strains of Porphyra yezoensis differing in their phycoerythrin(PE) content; the red strain, richer in PE than the wild strain,and the green and the yellow strains, poorer in PE. Specialattention was given to possible alteration of pigment systemin response to PE content, especially in the green and the yellowstrains. The relative quantum yields of Chi a fluorescence at–196°C and O² evolution were compared. Four strains commonly showed a low yield in pigment system II(PS II) fluorescence on Chl a excitation. The yield was as lowas in those algae in which PS II has only a small portion ofChl a as the light harvester. Measurement of O₂ evolution gavethe same results. Results indicate that the functional compositionof Chl a system remains unaltered in four strains with differentPE content. PS II in the green and the yellow strains reflectsa reduction in the size of light-harvesting components, suggestingthat pigmentation in these strains is fixed genetically as asun-type. ⁴ On leave from University of Washington, Seatle, Washington,U.S.A. (Received September 18, 1982; Accepted December 25, 1982)     

The plasminogen activator inhibitor-1 binding site in the kringle-2 domain of tissue-type plasminogen activator

We have shown that synthetic peptides containing the amino acid sequence Asn-Arg-Arg-Leu, derived from the amino acid sequence of the inner loop of the kringle-2 domain of tissue-type plasminogen activator (tPA), inhibited complex formation between two chain tPA and plasminogen activator inhibitor-1 (PAI-1) by binding to PAI-1. This binding was reversible and was inhibited by not only tPA but also by enzymatically inactive tPA. Quantitative analyses of the interaction of PAI-1 with the peptide containing the Asn-Arg-Arg-Leu sequence indicated that the PAI-1 binding site residues in the inner loop of the kringle-2 domain and is preferentially expressed in two chain tPA.     

The secondary electron acceptor of photosystem I in Gloeobacter violaceus PCC 7421 is menaquinone-4 that is synthesized by a unique but unknown pathway

The secondary electron acceptor of photosystem (PS) I in the cyanobacterium Gloeobacter violaceus PCC 7421 was identified as menaquinone-4 (MQ-4) by comparing high performance liquid chromatograms and absorption spectra with an authentic compound. The MQ-4 content was estimated to be two molecules per one molecule of chlorophyll (Chl) a', a constituent of P700. Comparative genomic analyses showed that six of eight men genes, encoding phylloquinone/MQ biosynthetic enzymes, are missing from the G. violaceus genome. Since G. violaceus clearly synthesizes MQ-4, the combined results indicate that this cyanobacterium must have a novel pathway for the synthesis of 1,4-dihydroxy-2-naphthoic acid.     

Minor but key chlorophylls in Photosystem II

A ‘metal-free’ chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.     

Identification of the primary electron donor in PS II of the Chl d-dominated cyanobacterium Acaryochloris marina

The primary electron donor of photosystem (PS) II in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina was confirmed by delayed fluorescence (DF) and further proved by pigment contents of cells grown under several light intensities. The DF was found only in the Chl a region, identical to Synechocystis sp. PCC 6803, and disappeared following heat treatment. Pigment analyses indicated that at least two Chl a molecules were present per each two pheophytin a molecules, and these Chl a molecules are assigned to P(D1) and P(D2). These findings clearly indicate that Chl a is required for water oxidation in PS II.     

Unique constitution of photosystem I with a novel subunit in the cyanobacterium Gloeobacter violaceus PCC 7421

Constitution of the photosystem I complex isolated from the cyanobacterium Gloeobacter violaceus PCC 7421 was investigated by tricine-urea-SDS-PAGE, followed by peptide mass fingerprinting or N-terminal sequencing. Eight subunits (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaL and PsaM) were identified as predicted from the genome sequence. A novel subunit (PsaZ) was discovered, but PsaI, PsaJ, PsaK and PsaX were absent. PsaB has a C-terminal extension with 155 amino acids in addition to the conserved region and this domain is similar to the peptidoglycan-binding domain. These results suggest that PS I complexes of G. violaceus have unique structural properties.     

Molecular organization of bacteriochlorophyll in chlorosomes of the green photosynthetic bacteriumChloroflexus aurantiacus: Studies of fluorescence depolarization accompanied by energy transfer processes

Examination was made of changes in fluorescence polarization plane by energy transfer in the chlorosomes of the green photosynthetic bacterium,Chloroflexus aurantiacus. Fluorescence anisotropy in the picosecond (ps) time region was analyzed using chlorosomes suspended in solution as well as those oriented in a polyacrylamide gel. When the main component of BChlc was preferentially excited, the decay of fluorescence anisotropy was found to depend on wavelength. In the chlorosome suspension, the anisotropy ratio of BChlc changed from 0.31 to 0.24 within 100 ps following excitation. In the baseplate BChla region, this ratio decreased to a negative value (–0.09) from the initial 0.14. In oriented samples, the degree of polarization remained at 0.68 for BChlc, and changed from 0.25 to –0.40 for the baseplate BChla by excitation light whose electric vector was parallel to the longest axis of chlorosomes. In the latter case, there was a shift from 0.30 to –0.55 by excitation perpendicular to the longest axis. Time-resolved fluorescence polarization spectra clearly indicated extensive changes in polarization plane accompanied by energy transfer. The directions of polarization plane of emission from oriented samples were mostly dependent on chlorosome orientation in the gel but not on that of the polarization plane of excitation light. Orientations of the dipole moment of fluorescence components was consistent with that of absorption components as determined by the linear dichroism (Matsuura et al. (1993) Photochem. Photobiol. 57: 92–97). A model for molecular organization of BChlc anda in chlorosomes is proposed based on anisotropic optical properties.     

Characterization of morphological conversion of Helicobacter pylori under anaerobic conditions

Helicobacter pylori (H. pylori), a gram‐negative microaerophilic bacterial pathogen that colonizes the stomachs of more than half of all humans, is linked to chronic gastritis, peptic ulcers and gastric cancer. Spiral‐shaped H. pylori undergo morphologic conversion to a viable but not culturable coccoid form when they transit from the microaerobic stomach into the anaerobic intestinal tract. However, little is known about the morphological and pathogenic characteristics of H. pylori under prolonged anaerobic conditions. In this study, scanning electron microscopy was used to document anaerobiosis‐induced morphological changes of H. pylori, from helical to coccoid to a newly defined fragmented form. Western blot analysis indicated that all three forms express certain pathogenic proteins, including the bacterial cytotoxin‐associated gene A (CagA), components of the cag‐Type IV secretion system (TFSS), the blood group antigen‐binding adhesin BabA, and UreA (an apoenzyme of urease), almost equally. Similar urease activities were also detected in all three forms of H. pylori. However, in contrast to the helical form, bacterial motility and TFSS activity were found to have been abrogated in the anaerobiosis‐induced coccoid and fragmented forms of H. pylori. Notably, it was demonstrated that some of the anaerobiosis‐induced fragmented state cells could be converted to proliferation‐competent helical bacteria in vitro. These results indicate that prolonged exposure to the anaerobic intestine may not eliminate the potential for H. pylori to revert to the helical pathogenic state.