Insect intestinal tracts harbor several novel, deep-rooting clades of as-yet-uncultivated bacteria whose biology is typically completely unknown. Here, we report the isolation of the first representative of the termite group 1 (TG1) phylum from sterile-filtered gut homogenates of a humivorous scarab beetle larva. Strain Pei191(T) is a mesophilic, obligately anaerobic ultramicrobacterium with a gram-negative cell envelope. Cells are typically rod shaped, but cultures are pleomorphic in all growth phases (0.3 to 2.5 mu m long and 0.17 to 0.3 mu m wide). The isolate grows heterotrophically on sugars and ferments D-galactose, D-glucose, D-fructose, D-glucosamine, and N-acetyl-D-glucosamine to acetate, ethanol, hydrogen, and alanine as major products but only if amino acids are present in the medium. PCR-based screening and comparative 16S rRNA gene sequence analysis revealed that strain Pei191T belongs to the "intestinal cluster," a lineage of hitherto uncultivated bacteria present in arthropod and mammalian gut systems. It is only distantly related to the previously described so-called "endomicrobia" lineage, which comprises mainly uncultivated endosymbionts of termite gut flagellates. We propose the name " Elusimicrobium minutum" gen. nov., sp. nov. (type strain, Pei191(T) = ATCC BAA-1559(T) = JCM 14958(T)) for the first isolate of this deep-branching lineage and the name " Elusimicrobia" phyl. nov. for the former TG1 phylum.

Phycobiliproteins (PBP) are major antenna pigments of cryptophytes, rhodophytes and cyanobacteria. According to their chemical structure and spectral properties four major classes of PBP can be distinguished: phycoerythrin (PE), phycoerythrocyanin (PEC), phycocyanin (PC) and allophycocyanin (AP). In cyanobacteria and red algae PBP are organized as phycobilisomes (PBS), large complexes (molecular mass 5000-30000 kDa) that are attached to photosystem II at the cytoplasmic or stromal side of the thylakoid membranes. They are normally constructed of three-cylindrical core units from which several peripheral rods radiate. The core cylinders contain AP while the peripheral rods are composed of PC, either alone or with PE or PEC. The basic unit of the biliproteins is a heterodimer composed of an α- and a β-subunit with molecular masses between 15 and 22 kDa. They are aggregated in trimers of the structure (αβ)3,which in turn form hexamers by a tight face-to-face-association. For a review on PBS structure, see [1]. In cryptophytes PBP are located in the thylakoid lumen. Here, their supramolecular organization is not yet fully understood.

The main chlorophyll a/c light harvesting complex of the diatom Cyclotella cryptica was isolated by sucrose density gradient centrifugation. It consisted of two polypeptides of Mrs 18000 and 22000. Both polypeptides and fragments thereof, obtained by formic acid treatment, were blocked at their N-termini. An antiserum raised against the two subunits selectively immunolabeled the thylakoid within the chloroplasts. The subunits were nuclear encoded and could be immunoprecipitated from poly (A)(+) RNA as precursor proteins in the Mr range of 20000 to 24000. The existence of minor chlorophyll protein complexes and their possible function in light climate adaptation processes was investigated in cells adapted to low light and high light conditions. Low light grown cells contained more fucoxanthin and less beta-carotene relative to chlorophyll a than high light adapted cells. The xanthophyll cycle pigments diatoxanthin and diadinoxanthin increased five-fold relative to chlorophyll a under high light conditions. Western-immunoblotting experiments with antisera raised against several chlorophyll a/b and chlorophyll a/c antenna complexes demonstrated that, beside the dominating chlorophyll a/c light harvesting complex, minor antenna complexes might exist, which, in part, seem to react to the light climate applied.

culturable numbers estimated at between 1.2 3 105 and 7.3 3 105 cells per g of dry soil. The average cell volumes of all three strains were 0.03 to 0.04 mm3, and therefore they can be termed ultramicrobacteria or "dwarf cells." The small cell size is a stable characteristic, even when the organisms grow at high substrate concentrations, and thus is not a starvation response. All three strains have genomic DNA with a mol% G1C ratio of about 63, are gram negative, and are motile by means of a single flagellum. The three new isolates utilizedonlysugarsandsomesugarpolymersassubstratesforgrowth.Themetabolismisstrictlyfermentative, but the new strains are oxygen tolerant. Sugars are metabolized to acetate, propionate, and succinate. Hydrogen production was not significant. In the presence of 0.2 atm of oxygen, the fermentation end products or ratios did not change. The phylogenetic analysis on the basis of 16S ribosomal DNA (rDNA) sequence comparisonsindicatesthatthenewisolatesbelongtoabranchoftheVerrucomicrobialeslineageandareclosely related to a cloned 16S rDNA sequence (PAD7) recovered from rice paddyfield soil from Japan. The isolation of these three strains belonging to the orderVerrucomicrobialesfrom a model rice paddy system, in which rice was grown in soil from an Italian rice field, provides some information on the possible physiology and phenotype of the organism represented by the cloned 16S rDNA sequence PAD7. The new isolates also extend our knowledge on the phenotypic and phylogenetic depths of members of the orderVerrucomicrobiales, to date acquired mainly from cloned 16S rDNA sequences from soils and other habitats.

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic alpha-helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC II of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC II and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or II. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.

Phycobilisomes are the major light-harvesting antennae of cyano-bacteria and red algae. They transfer the absorbed light energy with high efficiency to photosystem II (PSII). The phycobilisomes are bound to the external surface of the thylakoids where they are organized to well aligned arrays (1). They are bound to PSII, as was shown by the isolation of oxygen-evolving PSII-phycobilisome complexes (2). It was the aim of our studies to examine the structure and organization of PSII-phycobilisome complexes of cyanobacteria and red algae.

Particle frequency of the peribacteroid membrane (PBM) from nodules of Glycine max (L.) Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101 (wild-type strain) was determined by freeze-fracturing to be about 2200·μm-2 in the protoplasmic fracture face and 700·μm-2 in the exoplasmic fracture face. In membranes isolated from nodules infected with the mutant RH 31-Marburg of B. japonicum, the particle frequency was similar in both fracture faces with 1200–1300 particles·μm-2. Analysis of particlesize distribution on peribacteroid membranes showed a loss, especially of particle sizes larger than 11 nm, in the mutant-infected nodules. Two-dimensional gel electrophoresis (isoelectric focussing and sodium dodecyl sulfate-polyacrylamide) showed 27 different polypeptides in the PBM from nodules infected with the wild-type strain, four of which were absent from the PBM of nodules infected with the mutant RH 31-Marburg, which also exhibited one extra small-molecular-weight polypeptide. At least 14 of the 27 polypeptides in the PBM from the wild-type-infected nodule were glycoproteins. In three of these glycoproteins, post-translational modifications were either lacking or different when the membrane was derived from mutant-infected nodules.

The supramolecular structure of the exoplasmic freeze-fracture particles of thylakoids of the thermophilic cyanobacterium Synechococcus sp. is compared with that of isolated photosystem-II complexes. The in-situ EF particles are scattered on the thylakoids or organized in rows of variable length; the latter aligned particles measure 10 nmx20 nm and are separated perpendicular to their long axis into two parts. We propose that they represent dimers composed of two monomeric 10-nm EF particles side by side. Isolated photosystem (PS)II particles correspond in size to the monomeric 10-nm EF particles as analysed by negative contrast and freeze-fracture electron microscopy. Dimeric PSII particles, very similar to the in-situ 10 nmx20 nm EF particles, are obtained after incorporation of purified PSII complexes into liposomes made from phospholipid and cholesterol. Each monomeric complex consists of the reaction center, the water-splitting system, the chlorophyll antennae and phycobilisome-binding polypeptides. We propose that the dimeric complexes bind one hemidiscoidal phycobilisome at their domains exposed to the external side of the thylakoids. The implications of this arrangement of the PSII-phycobilisome complexes within the thylakoids upon excitation-energy distribution are discussed.

In nodules of Glycine max cv. Mandarin infected with a nod (+)fix(-) mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod(+) fix(-) mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.

Cytochrome f/b6 and CF0-CF1 ATP synthetase complexes from spinach chloroplasts were reconstituted into liposomes prepared from phospholipids and purified spinach galactolipids. Freeze-fracture analysis showed homogeneous particle populations spanning the lipid membranes with their elongated axes. The lipid composition of the liposomes had no effect on the size of the reconstituted complexes. The average diameter of cytochrome f/b6 measured 8.5 nm and of the CF0 part of the ATP-synthetase 9.6 nm. When reconstituted cytochrome f/b6 particles were crosslinked by specific antibodies on the liposomes, they formed hexagonal or square arrays with an 8.3 nm center to center particle spacing. Agglutination studies of inside out and right side out thylakoid vesicles revealed the antigenic determinants of cytochrome f polypeptides to be exposed on the inner thylakoid surface and to be present in stacked and unstacked membrane regions.

Phycobilisomes of red algae and cyanobacteria contain small amounts of nonpigmented polypeptides in addition to the major constituent biliprotein pigments. The localization of these polypeptides is analyzed by gel electrophoresis of phycobilisome fragments obtained by selective dissociation and subsequent separation. Five groups of biliprotein aggregates are determined, belonging to the 6, 11, 16, 18 and 23 S categories. Accessory nonpigmented high molecular weight proteins (80,000 MW) are exclusively bound to phycobilisome core fractions and thylakoids, thus apparently serving as links between the phycobilisomes and the photosynthetic units of the thylakoids. In contrast, smaller nonpigmented accessory polypeptides of 20,000 to 60,000 MW are preferably found in the peripheral biliprotein stacks. They may either form a compatible link between the phycobilisome core and periphery or bind and co-polymerize with hexameric biliproteins in the peripheral stacks to enhance or effect binding of the aggregates. Furthermore, they may determine the arrangement and composition of the phycobilisomes during development and chromatic adaptation.

Hexameric B-phycoerythrin (alpha beta)6 gamma is a double disc of about 10.7 x 4.3 nm; each single disc consists of a six membered periphery (alpha beta)3, the subunits of which are assumed to be associated in alternating positions with little or no staggering. A central subunit, almost certainly the gamma-subunit, penetrates both rings linking them tightly together. Hexameric C-phycocyanin (alpha beta)6 has the same construction but lacks this central subunit. In urea gel electrophoresis B-phycoerythrin I and II separate into three bands alpha, beta, and gamma in a relative molar ratio of 6:6:1. The molecular weights of the alpha-, beta-, gamma-subunits, estimated from SDS gels were 18 700, 18 700 and 29 200 and 18 300, 18 300 and 29 900 for B-phycoerythrin I and II, respectively, resulting in molecular weights of 253 600 and 249 500 for both hexameric aggregates. In density gradient centrifugation a sedimentation coefficient s20,w . 10(13) of 11.3 and a molecular weight of 244 000 were calculated. In sedimentation analyses of partially dissociated phycobilisomes a fragment consisting of two phycoerythrin hexamers with a sedimentation constant of 18 S (dodecamer) and tripartite units with two B-phycoerythrin hexamers associated with one polar C-phycocyanin hexamer with a sedimentation constant of 22 S were demonstrated. The corresponding molecular weight of the tripartite units, about 800 000, coincides well with morphological measurements on the basis of an average protein packing density and with earlier estimates on cross-linked biliprotein aggregates in gradient gel electrophoresis. The spaces of 1.2 to 3.0 nm between the hexamers give rise to a strong 6.0 nm periodicity within the tripartite units, the weak 3.0 nm periodicity originates from the double-rings of the constituent hexamers.

Bacteroids in ineffective (nitrogenase negative) nodules of Glycine max, infected with Rhizobium japonicum 61-A-24, as compared to those in effective nodules are characterized by reduced specific activities of alanine dehydrogenase to 15%, of 3-hydroxybutyrate dehydrogenase to 50%, and an increase of glutamine synthetase to 400%. In the plant cytoplasm of ineffective nodules, glutamine synthetase activity is reduced to 10–30%, glutamate dehydrogenase to 50–70%, and the aspartate aminotransferase and alanine aminotransferase are enhanced to 120–200%, depending on the age of the nodules. The total pool of soluble amino acids is reduced to 52 μmol per g nodule fresh weight, as compared to 186 μmol in effective nodules, with a replacement of asparagine (42 mol% of the amino acids) by an unknown amino compound. This compound is absent in nitrogenase, repressed and derepressed, free-living Rhizobium japonicum cells and in the uninfected root tissue. In nitrogenase derepressed, as compared to the repressed free-living cells of Rhizobium japonicum 61-A-101, arginine shows the most obvious change with a reduction to less than one tenth. The ultrastructure of the ineffective nodule is different from the effective organ even in the early stages. The membrane envelopes of the infection vacuoles are decomposing in heavily infected cells within 18 to 20 d after infection. In lightly infected cells very large vacuoles develop with only a few bacteroids inside. No close associations of cristae-rich mitochondria with amyloplasts are observed as in effective nodules. The uninfected cells keep their large starch granules even 40 d after infection. Some poly-β-hydroxybutyrate accumulation in the bacteroids is observed but only in the early stages, and it is almost absent in old nodules (40 d). At this age the infected cells are obviously compressed by uninfected cells, whereas in effective nodules with nitrogenase activity and leghaemoglobin formation, the infected cells have a much higher osmotic pressure than the neighbouring uninfected cells.

ZusammenfassungDie Biliproteide der Cryptophyceen Cryptomonas maculata (Phycoerythrin‐545), Chroomonas spec. (Phycocyanin‐645) und Hemiselmis rufescens (Phytoerythrin‐555) lassen sich in der Aggregationsform von Dimeren (αβ)2 isolieren.In Ultradünnschnitten strukturell gut erhaltener Zellen sind sie entweder dispers innerhalb der Lumina der Thylakoide vorhanden oder lokal in Form feiner Säulenreihen senkrecht zur inneren Oberfläche der Thylakoidmembranen ausgerichtet.Die Biliproteide B‐Phycoerythrin, C‐Phycocyanin und Allophycocyanin der einzelligen Rotalge Rhodeila violacea sind in vivo als Phycobilisomen aggregiert und mit der äußeren Oberfläche der Thylakoidmembranen verbunden.Bei stärkerer Vergrößerung bestehen isoliertes natives C‐Phycocyanin und Allophycocyanin aus sechs globulären Proteinuntereinheiten in ringförmiger Verknüpfung mit einem zentralen Loch. Diese Aggregate können als Trimere (αß)3 angesehen werden. Natives B‐Phycoerythrin auf der anderen Seite enthält neben der ähnlich aufgebauten Peripherie eine zentrale Untereinheit, die zwei flächig miteinander aggregierte Ringe globulärer Proteinuntereinheiten zusammenhält.Dreiereinheiten, ein gut definiertes Bauelement der Phycobilisomenperipherie von Rhodella violaceae, enthalten B‐Phycoerythrin und C‐Phycocyanin im molaren Verhältnis 2: 1 (koller et al. 1978). Jedes der drei Scheibchen der Dreiereinheit besitzt eine ähnliche Morphologie in Profilansicht und besteht aus zwei Ringen globulärer Proteinuntereinheiten. Diese Tatsache steht in Einklang mit den Beobachtungen an Isolaten nativen B‐Phycoerythrins und stützt die Ansicht einer flächigen Aggregation von zwei trimeren C‐Phycocyanin‐Ringen in vivo zum Aufbau eines Teils der Dreiereinheit.Ein verbessertes Modell des Phycobilisoms von Rhodella violacea wird vorgestellt.

Heterogeneous complexes with a molecular weight of about 790000 containing B-phycoerythrin (Bangiales phycoerythrin) and C-phycocyanin (Cyanophyceae phycocyanin) in a molar pigment ratio of 2:1 were isolated from purified, dissociated phycobilisomes. Electron microscopical investigations revealed structures of three discs aggregated face to face with an apparent distance of 1.5 nm between each disc. Two discs may represent phycoerythrin and one phycocyanin. The complexes are structurally identical with tripartite units of the phycobilisome periphery. Fluorescence data confirmed the integrity of isolated tripartite units. Excitation at 546 nm gives a fluorescence maximum at 644 nm, indicating intermolecular transfer of excitation energy from phycoerythrin to phycocyanin. Comparative subunit analyses and spectral data suggested that no allophycocyanin is present. Cross-linking experiments gave evidence for a polar arrangement of phycocyanin within the complex. This pigment itself is an aggregate of two smaller molecules each having a molecular weight of about 140000. Tripartite units contain all the phycoerythrin and phycocyanin of the phycobilisome. On this basis, a phycobilisome model is proposed which combines the aspects of biliprotein distribution, energy transfer and fine structure.

Plants of Glycine max var. Caloria, infected as 14 d old seedlings with a defined titre of Rhizobium japonicum 3Il b85 in a 10 min inoculation test, develop a sharp maximum of nitrogenase activity between 17 and 25 d after infection. This maximum (14±3 nmol C2H4 h-1 mg nodule fresh weight-1), expressed as per mg nodule or per plant is followed by a 15 d period of reduced nitrogen fixation (20–30% of peak activity). 11 d after infection the first bacteroids develop as single cells inside infection vacuoles in the plant cells, close to the cell wall and infection threads. As a cytological marker for peak multiplication of bacteroids and for peak N2-fixation a few days later the association of a special type of nodule mitochondria with amyloplasts is described. 20 d after inoculation, more than 80% of the volume of infected plant cells is occupied by infection vacuoles, mostly containing only one bacteroid. The storage of poly-β-hydroxybutyrate starts to accumulate at both ends of the bacteroids. Non infected plant cells are squeezed between infected cells (25d), with infection vacuoles containing now more than two (up to five) bacteroids per section. Bacteroid development including a membrane envelope is also observed in the intercellular space between plant cells. 35 d after infection, more than 50% of the bacteroid volume is occupied by poly-β-hydroxybutyrate. The ultrastructural differentiation is discussed in relation to some enzymatic data in bacteroids and plant cell cytoplasm during nodule development.

Three multiple phycoerythrin-545 forms were purified from crude extracts of Cryptomonas maculata by preparative isoelectric focusing. The phycoerythrin forms are charge isomers with isoelectric points at 7.83, 5.05 and 4.84. The multiple pigment forms have similar molecular weights of 44500 daltons and are composed of subunits of unequal size in a 1:1 stoichiometry with molecular weights of (α) 9900 and (β) 15700 daltons, twice. The proposed quarternary structure of the native pigments is (α)2(β)2.

The properties of phycocyanin-645 from the fresh water cryptomonad Chroomonas spec. were investigated after the pigment was isolated and purified by a combination of differential ammonium sulphate fractionation, gel filtration chromatography and ammonium sulphate gradient elution.