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Viren sind obligate Parasiten und deshalb fur ihre Vermehrung auf die lebenden Zellen ihres Wirtes angewiesen. Bevor dieser stirbt, mussen die Viren einen neuen Wirt infiziert haben, damit sie uberleben konnen.

Die Taxonomie von Viren war anfangs uneinheitlich. Es existierten mehrere Konzepte, von denen jedoch keines von allen Virologen akzeptiert wurde. Ein allgemein anerkanntes System der Nomenklatur konnte nur durch Kooperation auf internationaler Basis entstehen. Die Grundlagen fur eine einheitliche Klassifizierung wurden 1966 auf einer Konferenz in Moskau diskutiert und das International Committee on Taxonomy of Viruses (ICTV) gegrundet, das seit 1971 regelmasig Berichte uber Virustaxonomie veroffentlicht, die die Einordnung von Viren verbindlich festlegen. Jede Klassifizierung der Viren sollte neben der Phylogenie (der evolutionaren Geschichte) phanotypische (praktische Charaktermerkmale) berucksichtigen. In dem neuesten Bericht (van Regenmortel et al. 2000) sind bei den Pflanzenviren 977 Spezies aufgefuhrt. Eine formale Definition einer Spezies wurde 1991 von der ICTV gegeben: A virus species is a polythetic class of viruses that constitute a replicating lineage and occupy a particular ecological niche.

Viruserkrankungen von Kulturpflanzenbestanden werden meist von Ausenstehenden als wenig besorgniserregend beurteilt beziehungsweise gar nicht als Erkrankung erkannt, da in vielen Fallen die Symptome an den Handelsprodukten nicht unbedingt als Schaden auffallen. Im Bestand selbst wird, im Gegensatz zu tierischen oder humanen Virosen, ein einzelnes befallenes Individuum kaum zur Kenntnis genommen. Erst bei Ausfall kompletter Bestande wird ein Virusbefall ernst genommen. Dies ist dann umso ernster, als dem Praktiker im Gegensatz zu pilzlichen Erkrankungen keinerlei direkte kurative Bekampfungsmasnahmen in Form von chemischen oder biologischen Pflanzenschutzmitteln zur Verfugung stehen. Zwar gab es auch im Pflanzenbereich Untersuchungen zum Einsatz von Nukleotidanaloga als Viruzide (Lerch 1987). Unter der derzeit kritischen Einschatzung des chemischen Pflanzenschutzes und den damit verbundenen Risiken fur Natur und Umwelt sind solche Verfahren neben dem immensen Kostenfaktor jedoch kaum als einsatzwurdig und akzeptabel anzusehen.

Viroide sind nackte, kovalent geschlossene, autonom in einer Wirtszelle replizierende, kleine RNA-Molekule, die keine Proteine kodieren. Der Begriff Viroid wurde von Diener (1971) bei der Beschreibung des Potato spindle tuber viroids eingefuhrt. Satellitenviren sind isometrische, mit Capsid ausgestattete Viren, die ihr eigenes Capsidprotein kodieren. Ihre Vermehrung ist auf ein Helfervirus angewiesen, das unabhangig vom Satellit Virus infizieren und replizieren kann (A-Typ). Satelliten-RNAs oder DNAs sind kleine Nukleinsauren, deren Enkapsidierung und Vermehrung von Helferviren abhangig sind. Sie konnen Proteine kodieren (B-Typ) oder ihnen fehlt eine messenger-Funktion (C- und D-Typ). Letztere sind unter 0,7 kb gros. Die Sequenz der Satellitennukleinsaure unterscheidet sich von der der Helfer. Defekte RNAs und hullproteinabhangige Replikons werden in diesem Kapitel nicht behandelt. Nahere Informationen konnen aus Bruening (2001) entnommen werden.

In einem Bericht aus dem 8. Jahrhundert wird die Adernvergilbung bei Eupatorium chinense erwahnt. Uber schwere Ernteschaden an Kulturpflanzen wie Ruben, Kassava und Mais wird seit uber 100 Jahren berichtet. Diese Erkrankungen werden durch Geminiviren verursacht. Aber erst in den 1970er-Jahren konnten die Viren, die diese Krankheiten auslosen, nachgewiesen und naher charakterisiert werden (Buck 1999).

Zu den ssRNA-Viren, deren Partikelmorphologie mit „starre Stabchen“ beschrieben wird, zahlt der Genus Tobamovirus (monopartite) und die Genera Benyvirus, Furovirus, Hordeivirus, Pecluvirus, Pomovirus, und Tobravirus (bi- oder multipartite; van Regenmortel et al. 2000). In diesem Kapitel wird primar auf die Eigenschaften von Mitgliedern der Genera Tobamo- und Tobravirus eingegangen, da diese sowohl in Bezug auf die Funktion ihrer Proteine als auch deren Interaktion mit den Wirtsproteinen am besten untersucht sind. Speziell interessierte Leser werden auf die systematischen Kapitel in diesem Buch (s. Kap. 6) oder auf die Arbeiten des ICTV und seiner Mitglieder verwiesen (Martelli 1997; van Regenmortel et al. 2000; Tidona u. Darai 2001).

Da Viren und subvirale Pathogene grundsatzlich bei ihrer Vermehrung auf (s. Kap. 9 bis 16) aufgefuhrt und ausfuhrlicher erklart.die Stoffwechselfunktionen lebender Wirtszellen angewiesen sind, haben sie im Laufe der Koevolution mit ihren Wirten die unterschiedlichsten Strategien entwickelt, um den Abwehrmechanismen der Wirte zu entgehen und die eigene Vermehrung zu erreichen. Dies gilt grundsatzlich fur alle Viren, egal welche Wirte sie befallen. Bei Pflanzenviren sind zusatzliche pflanzenspezifische Komponenten hinzugekommen, die den besonderen Eigenschaften ihrer Wirte gerecht werden, wie die Behinderung der Infektion und Ausbreitung im Wirt durch Zellwande und schutzende Abschlussgewebe sowie die Unbeweglichkeit der Pflanzen. In diesem Kapitel sollen, dem Fortgang einer Virusinfektion folgend, die einzelnen Schritte einer Virusinfektion beschrieben werden. Die speziellen Aspekte der Biologie der einzelnen Taxa der Viren werden im nachfolgenden Beispielteil (s. Kap. 9 bis 16) aufgefuhrt und ausfuhrlicher erklart.

Zu den Pflanzenviren mit einzelstrangigem negativem Sinn RNA-Genom (-ssRNA) gehoren mittlerweile funf Genera: Nucleo- und Cytorhabdovirus (Familie Rhabdoviridae), mit ungeteiltem Genom; Tospovirus (Familie Bunyaviridae), Tenuivirus und Ophiovirus mit drei- bzw. viergeteiltem Genom. Da das Genus Ophiovirus nur wenig untersucht ist, beschranken wir uns auf die Familie der Rhabdoviridae und die Genera Tospo- und Tenuivirus.

Retro- und Pararetroviren kopieren in einem bestimmten Entwicklungsstadium die genomische Information in den anderen Typ an Nukleinsaure. Bei den Retroviren wird das virale RNA-Genom in DNA und bei den Pararetroviren das virale dsDNA-Genom in RNA mit Hilfe einer viruskodierten reversen Transkriptase transkribiert. Retrovirus-DNA wird obligatorisch mit einer viralen Integrase in das Wirtsgenom integriert. Bei Pflanzen sind bisher keine Retroviren, sondern nur Pararetroviren bekannt geworden, die in der Familie der Caulimoviridae zusammengefasst werden. Pararetroviren, die Saugetiere infizieren, sind die Hepadnaviren.

Fur die Identifizierung von Viren in Feldproben sowie zur molekularen, strukturellen und biologischen Charakterisierung werden in der Pflanzenvirologie verschiedene Methoden eingesetzt. Dafur wurden einige gangige Methoden der Biochemie, Physiologie und Molekularbiologie modifiziert. Viele davon werden in den einschlagigen Lehrbuchern und Fachbuchern beschrieben. Andere Methoden, wie eine Infektion von Pflanzenmaterial oder die Reindarstellung von Viruspartikeln, sind dagegen nur in der Pflanzenvirologie zu finden. Grundsatzlich gibt es zum Nachweis von Viren verschiedene Ansatze. So kann in einem Biotest die Infektiositat eines Virus gezeigt werden, das Elektronenmikroskop gibt die Form des Partikels wieder, die ein wesentliches Merkmal fur die taxonomische Zuordnung darstellt. Andere Methoden beruhen auf dem Nachweis von viralen Proteinen oder Nukleinsauren. Um ein Virus eindeutig zu charakterisieren, sollten verschiedene Eigenschaften untersucht werden. Die eindeutige Zuordnung einer Krankheit zu einem Erreger ist nur nach Erfullen der Koch-Postulate moglich (s. Kap. 1.1).

Pflanzenviren mit flexiblen Stabchen als Partikelmorphologie sind nur bei den (+)ssRNA-Viren vertreten. Sie sind typisch fur folgende Taxa: Potyviridae, Closteroviridae, Capillovirus, Trichovirus, Vitivirus, Carlavirus, Potexvirus, Allexivirus und Foveavirus. Zu den taxonomischen Eigenschaften der einzelnen Taxa s. Kap. 6. Die Potyviridae stellen mit 198 Spezies die bei weitem groste Gruppe von allen Pflanzenviren dar. Allein das Genus Potyvirus besteht aus 180 Arten. Da von diesen Erregern bedeutende Kulturpflanzen befallen werden, wurden sie sehr fruh untersucht und eine Fulle von Informationen zusammengetragen. Deshalb soll diese Gruppe stellvertretend fur andere Taxa mit gleicher Struktur in diesem Kapitel abgehandelt werden. Weitergehende Informationen zur Familie der Potyviridae sind in Shukla et al. (1994) und Tidona u. Darai (2001) zu finden.

Die Pflanzenviren mit isometrischen Partikeln und einer ss(+)RNA als Genom werden grostenteils sechs Familien, namlich den Bromoviridae, Comoviridae, Luteoviridae, Sequiviridae, Tombusviridae und Tymoviridae, zugeordnet. Daneben gibt es noch einige nicht zugeordnete freie Genera, wie z. B. Marafivirus, Sobemovirus und Umbravirus (s. Kap. 6.5). Die Genome sind entweder ungeteilt oder bestehen aus zwei (Comoviridae) bzw. drei RNA-Segmenten (Bromoviridae). Unabhangig von der zum Teil sehr ahnlichen Partikelmorphologie und der Verteilung der genetischen Information auf ein bis mehrere RNA-Segmente konnen die Taxa drei verschiedenen Superfamilien zugeordnet werden. Zur picornaahnlichen Supergruppe, die sich durch posttranslationales Prozessieren der Translationsprodukte der monocistronischen RNA-Segmente, 5'-standige VPgs und polyA-Sequenzen am 3'-Ende auszeichnet, gehoren die Sequiund die Comoviridae. Dagegen konnen die Bromo- und Tombusviridae zur alphaahnlichen Supergruppe gerechnet werden, die sich durch gemeinsame Sequenzmotive im Bereich der replikationsbeteiligten Proteine auszeichnen. Eine eigene Stellung nehmen die Luteoviridae ein, die aufgrund von Ahnlichkeiten der replikativen Proteine entweder mit den Tombusviridae (Luteovirus) oder dem Genus Sobemovirus (Polerovirus) verwandt sind (Goldbach u. Wellink 1988).

In der Entdeckungsphase wurden Viren als „infektiose, filtrierbare Agenzien“ beschrieben. Wie der kurze, geschichtliche Ruckblick zeigt, konnte die Natur der Viren in dieser Zeit nicht aufgeklart werden. Der Begriff Virus, wie wir ihn heute verstehen, entwickelte sich aus dem zunehmenden Wissen uber Struktur, Organisation und Vermehrung der Viren. Die geordnete Struktur der Viren und ihre Zusammensetzung aus Protein(en) und einer Nukleinsaure wurde nachgewiesen und man erkannte, dass eine zellulare Organisation fehlt. Die Viren verfugen uber keinen eigenen Energieund Baustoffwechsel und die Vermehrung ist von einer intakten und funktionsfahigen Wirtszelle abhangig. Viren sind also keine Organismen, besitzen aber eine genetische Information fur ihre identische Vermehrung.

Die Genome von Viren sind im Vergleich mit organismischen Genomen klein. Dieses Wissen und die Tatsache, dass die Masse eines Proteins nur einen kleinen Teil der Masse des kodierenden Gens ausmacht, fuhrten Crick u. Watson (1956) zu der Annahme, dass das Capsid, die Proteinhulle der Viren, aus multiplen Kopien eines oder weniger Genprodukte aufgebaut ist. Sie machten auch die Voraussage, dass die gleichen Untereinheiten durch identische Kontakte mit ihren Nachbarn verbunden sind und so symmetrische Strukturen bilden. Die Packung asymmetrischer Proteine in regelmasig strukturierte Partikel kann nach den Prinzipien helikaler oder kubischer Symmetrie erfolgen. Von allen Formen kubischer Symmetrie erlaubt das Ikosaeder die Bildung der grosten Strukturen bei gegebener Grose der Untereinheiten. Caspar u. Klug (1962) konnten durch ihre elektronenmikroskopischen Untersuchungen diese Symmetrieformen nachweisen, die offensichtlich wahrend der Evolution fur den Aufbau der Capside selektioniert wurden. Es entstanden auch komplexere Strukturen, die aber in der Regel auf eine der beiden Symmetrieformen zuruckgefuhrt werden konnen (Abb. 4.1).

Most of the bacterial photosynthetic reaction centres known to date contain a cytochrome subunit with four covalently bound haem groups. In the case of Blastochloris viridis, this reaction centre subunit is anchored in the membrane by a lipid molecule covalently attached to the cysteine which forms the N-terminus of the mature protein after processing by a signal peptidase. We show that posttranslational N-terminal cleavage of the cytochrome subunit does not occur in the aerobic photosynthetic bacterium Roseobacter denitrificans. From sequence analysis of the resulting elongated N-terminus it follows that a transmembrane helix is anchoring the reaction centre-bound cytochrome in the membrane. Comparative sequence analysis strongly suggests that all cytochrome subunits lacking the lipid coupling cysteine share this structural feature. Comparison of the N-terminal segment of the cytochrome subunit of Roseobacter denitrificans with the sequences of the PufX proteins from Rhodobacter sphaeroides and Rhodobacter capsulatus suggests a phylogenetic relation.

Publisher Summary Techniques of molecular genetics enable scientists to investigate the development of organisms, the differentiation of tissues, and their regulation at the molecular level. Anton de Bary discovered the sequence of sexual and asexual propagation of fungi by following the different stages of the development from the spore to fructification organs through observation with the microscope in vitro and in vivo after inoculation of plants with spores. De Bary's second great achievement was his major contribution to phytopathology. Anton de Bary's observation and experimental results were explained in the frame of a unifying concept of the phylogeny of fungi, where numerous studies on algae, lichens, ferns, and higher plants enriched the knowledge of his time. The chemical composition, basic structure and function of the major cellular constituents, at least of model organisms, are known at present.

The puc operon of Rhodobacter capsulatus consists of the genes pucBACDE. The genes pucB and pucA encode the pigment-binding polypeptides LHIIβ and LHIIα and pucC encodes a regulatory protein, essential for the expression of the LHII complex. The function of the gene product PucD is unknown; pucE encodes the γ polypeptide of the LHII peripheral light-harvesting complex. The LHII complex is formed in variable amounts mainly under the control of the light intensity (1-3). PucC is a membrane-bound protein which spans the membrane presumably 12 times. The C terminal transmembrane segments are important for the function (4). Mutation of pucC by transposon insertion or deletion results in a strong reduction of puc mRNA (2, 5) and a complete inhibition of the formation of the LHII complex (1-4). The gene products of pucDE seem to stabilize the LHII complex (2) and their absence inhibits growth (3). For the investigation of the puc operon the mutant strain NK3 of Rb. capsulatus was used up to now. This strain contains a Tn5 insertion in pucC (1, 2, 5) which did not suppress completely the expression of the puc operon but has a polar effect on transcription. Therefore, strains were constructed which have chromosomal deletions in pucBACDE, pucDE and pucD, respectively. The expression of puc genes in these and plasmid-reconstituted strains was investigated.

Photosynthesis was discovered by its metabolic aspect, the CO2 assimilation and the O2 production of green plants as a light-dependent process catalyzed by chlorophyll (J. Priestley 1772, J. Ingenhousz 1779, N. T. de Saussure, 1804, H. Dutrochet 1837). Justus Liebig demonstrated 1840 that plants can grow on pure inorganic nutrients (HCO3 -, NH4 +, SO4 2+ and trace elements). The law of maintenance of energy and the unit of heat (calorie) were discovered by J. Robert Mayer (1845), J. P. Joule (1818-1889) and Hermann L. Helmholtz (1821-1894), at the middle of the century, but the energetic aspect of photosynthesis, i.e. the transformation of light energy into chemical energy, was recognized many decades after demonstration of CO2 fixation. Wilhelm Engelmann wrote 1888 “The purple schizomycetes (bacteria) step in the group of organisms which assimilate like green plants. The bacteriopurpurin (bacteriochlorophyll) is a true chromophyll, so far the actual absorbed light energy is transformed in potential chemical energy”.

Bacteriology paved the way to cell biology - a historical account the procaryotic cell basic prerequisites for cellular life diversity of metabolic pathways the genetics of the prokaryotes and their viruses gene expression and regulatory mechanism cell growth and differentiation diversity and systematics prokaryotes in the biosphere applied microbiology.

In Rhodobacter capsulatus B10 (wild type) both alpha subunits of the light-harvesting complexes are phosphorylated during photosynthetic membrane synthesis. During the process of insertion of these polypeptides, there is a dephosphorylation resulting in intracytoplasmic membranes in which no radioactive phosphate could be detected. Moreover, we show that their phosphate-specific contents depends on the growth conditions, the highest being observed under oxic conditions. When photosynthetic membrane synthesis was induced under light and anaerobiosis, a decrease in the phosphate-specific contents ensued. An inverse relationship exists between specific phosphorylation levels and the degree of membrane differentiation. The phosphorylation is thus a transient phenomenon characteristic of the photosynthetic membrane synthesis governed by the external redox conditions constituting an additional post-transcriptional level of regulation.

The activities of NAD+-photoreduction and NADH/decyl-ubiquinone reductase in membrane preparations of Rhodobacter capsulatus changed to the same extent under different conditions. These results indicated that NADH:ubiquinone oxidoreductase (complex I) catalyzes the electron transport in the downhill direction (respiratory chain) and in the uphill direction (reverted electron flow). This conclusion was confirmed by the characterization of a complex-I-deficient mutant of R. capsulatus. The mutant was not able to reduce NAD+ in the light. Since this mutant was not able to grow photoautotrophically, we concluded that complex I is the enzyme that catalyzes the reverted electron flow to NAD+ to provide reduction equivalents for CO2 fixation. Complex I is not essential for the reverted electron flow to nitrogenase since the mutant grew under nitrogen-fixing conditions. As shown by immunological means, NuoE, a subunit of complex I from R. capsulatus having an extended C-terminus, was modified depending on the nitrogen source present in the growth medium. When the organism used N2 instead of NH4 +, a smaller NuoE polypeptide was synthesized. The complex-I-deficient mutant was not able to modify NuoE. The function of the modification is discussed.

Roseobacter (Rs.) denitrificans belongs to the so-called aerobic photosynthetic bacteria. This type of bacteria is closely related to purple bacteria, but in contrast to these ‘true’ photosynthetic bacteria, they are not able to grow under anoxic conditions (1). Photosynthetic protein complexes and bacteriochlorophyll are only built under oxic conditions in these bacteria. Rs. denitrificans is phylogenetically close to Rhodobacter (Rb.) capsulatus a true phototrophic purple bacterium (2). The puf operon encoding the protein subunits L, M, C of the reaction center (RC) and of light harvesting complex I (LHI) from Rs. denitrificans were cloned and sequenced and show very high similarities to purple bacteria in either single sequences or the whole operon structure (3). Despite these similarities charge separation does not occur in the reaction center under anoxic conditions (4). The lack of photosynthetic activity was believed to be caused by the overreduction of the electron transfer system (1) and a higher midpoint redox potential of the primary acceptor (QA) of the RC in aerobic phototrophs compared to that of typical purple bacteria (5). For a better understanding of the charge separation in Rs. denitrificans RC the M and L subunits of RC from Rs. denitrificans were combined with the H-subunit from Rb. capsulatus in a Rb. capsulatus puf puc negative mutant (6). Charge recombination between the primary donor P3+ and Q A − was observed in the transconjugant indicating a correct assembly of L and M subunits but the transconjugant did not grow under anoxic conditions. In Rhodobacter sphaeroides it was shown that the H-subunit has an influence on electron transfer from QA to QB (7, 8). Since the H-subunit could be responsible for the differences between purple bacteria and aerobic phototrophs we cloned and sequenced the RC H-subunit from Rs. denitrificans. To get informations about the interaction between the quinones and the surrounding protein matrix a model of the HLM-complex of the RC was calculated.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.

The light-harvesting (LH) complex I (B870) of anoxygenic photosynthetic purple bacteria is the oligomeric form of its subunit B820 consisting of the low-molecular-weight polypeptides alpha, beta, bacteriochlorophyll (BChl), and carotenoids in the stoichiometric ratio [alpha1 beta1 (BChl2) Crt1-2]n. LHI surrounds the photochemical reaction center (RC). The major absorption band of the LHI complex is species-specific and is found at 870-890 nm; those of the subunit and the monomeric BChl a (dissolved in methanol) absorb at 820 and 770 nm, respectively. The isolated LHI complex can be reversibly dissociated to the B820 subunit or to the polypeptides and pigments by addition of detergents. Reconstitution of the B820 or the functional B870 complex is still possible after partial truncation of the N- or C-terminal regions of the alpha- or beta-polypeptide or of the beta-polypeptide only. The minimal structural requirements for reconstitution of a spectrally wild-type form after truncation of the polypeptides and/or modifications of the BChl molecule are described. The insertion of the LHIalpha- and LHIbeta-polypeptides into the membrane and the in vivo assembly of LHI, studied in a cell-free system and in whole cells of Rhodobacter capsulatus, depend on the primary structures of both polypeptides, BChl, the chaperones DnaK and GroEL, membrane-bound proteins, and energized membranes. Exchanges, deletions, or insertions of amino acyl residues, especially in the conserved region of the N-terminus of the LHIalpha-polypeptide, prevent or reduce the efficiency and stability of the LHI assembly. Therefore, reconstitution of LHI in a detergent micelle does not exactly reproduce the formation of the LHI complex in the photosynthetic membrane in vivo. The N-terminal domains play a crucial role in the formation of the oligomeric protein scaffold and of the pigment array. Facultatively phototrophic bacteria such as Rhodospirillum (Rsp.) rubrum or Rhodobacter (Rba.) capsulatus can adjust to changes in oxygen tension, light intensity, temperature, and substrates to grow under chemotrophic or phototrophic conditions. The photosynthetic apparatus (PSA), localized mainly on intracytoplasmic membranes (ICM), is usually synthesized only under low oxygen partial pressure. The cellular amount and composition of the PSA are modified upon changing light intensity in relation to cell growth (Drews and Golecki 1995). The morphogenesis of cellular structures like ICM is quite different from self-assembly. Self-assembly is a reversible process of aggregation of the constituents of a complex structure without protein synthesis and is driven by weak or strong forces in the interactions of the constituents. Morphogenesis results from the interplay of numerous gene products and the cellular organization and is always dependent upon pre-existent structures (Harold 1995). The morphogenesis of the photosynthetic membrane in purple bacteria has been studied in its different steps. The regulation at the transcriptional and post-transcriptional levels in purple bacteria, and the structure and morphogenesis of the ICM have been described recently (Armstrong 1995; Bauer 1995; Biel 1995; Drews and Golecki 1995; Klug 1995). In this mini-review, I will focus on the minimal requirements for the in vitro assembly of light-harvesting (LH) complex I (B870) from its constituents in detergent micelles and compare the results with observations on the complex process of targeting and import of LHI polypeptides into the membrane and assembly of B870.

A respiring culture of Rhodobacter sphaeroides, grown in the dark under defined aerobic conditions, produced cells capable of immediately commencing adaptation to photosynthetic growth on exposure to light and further reduction of oxygen tension. Adaptation was complete after 12 h and the bacteriochlorophyll a content increased 10–20-fold. This adaptation was performed in the presence of either H218O or 18O2. The extracted bacteriochlorophyll a was examined by mass spectrometry to determine the origin of both the 3-acetyl and 131-oxo oxygen atoms: both were derived from water. The derivation of the 131-oxo group from water in R. sphaeroides indicates that the formation of isocyclic ring E from the 13-propionic acid methylester side chain of Mg2+-protoporphyrin IX mono-methylester is an anaerobic process involving a hydratase. This is very different to the situation in higher plants and green algae where the formation of isocyclic ring E is an aerobic process in which the 131-oxo group is derived from molecular oxygen via an oxygenase. In contrast to adapting R. sphaeroides cells, the 131-oxo group of bacteriochlorophyll a in growing cells of the obligate aerobic chemotrophic bacterium Roseobacter denitrificans, was labelled by 18O2 and is, therefore, derived from molecular oxygen like in higher plants and green algae; however, the 3-acetyl group was not labelled by 18O2. Thus, while the 131-oxo group has different origins in R. sphaeroides and R. denitrificans, the 3-acetyl group arises in both bacteria by enzymic hydration of the vinyl group of a chlorophyll a derivative.

Transcripts of the genes pufBA, pufB or pufA from Rhodobacter capsulatus were translated in a cell-free system of R. capsulatus. The incorporation of the nascent polypeptides LHIα and β in various types of membranes and the assembly of the light-harvesting (LH) complex I (B870) were investigated. The highest rate of stable incorporation of LHIα and β into the membrane was observed with membranes from the wild type strain grown under chemotrophic conditions. Addition of membranes from cells defective in biosynthesis of pigment-binding proteins resulted in a less efficient or less stable incorporation of LHIαβ. The single polypeptides LHIα or β were synthesized and inserted into the membrane but were extractable to a higher percentage by 6 M urea than the pairwise inserted LHI polypeptides. If the ribosomes and the S135 extract were depleted of DnaK the rate of synthesis of both polypeptides, LHIα and β, was strongly reduced. Removal of GroEL from the cell-free system did not impair the synthesis and membrane association of both proteins, but affected the stable insertion. A high percentage of the LHIαβ polypeptides became extractable by 6 M urea if the cell-free system was depleted of GroEL. Addition of GroEL to the cell-free system restored the capacity of stable insertion of both proteins into the membrane. GroEL interacted with LHIα and β before membrane targeting as shown by immunological means. A protein fraction, which can be removed from the membrane with a low-salt buffer, supported the effective and stable incorporation of LHIαβ into the membrane. It is concluded that the assembly of the LHI complex in the membrane system of R. capsulatus is a multistep process guided and supported by polypeptides located in the cytoplasm and in the membrane. In the cell-free in vitro system not only the correct insertion of the LHI polypeptides but also an assembly with bacteriochlorophyll was observed. BChl was synthesized from δ-amino levulinate in the cell free system.

The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800-850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 micrograms Bchl per mg cell protein when grown at high light intensity (20,000 lux) to 60 micrograms Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographic analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of alpha and one type of beta polypeptide in the purified complex, of which the N-terminal sequences have been determined.

We analyzed the 16S ribosomal DNAs of three obligately aerobic, bacteriochlorophyll a-containing bacteria, "Roseococcus thiosulfatophilus," "Erythromicrobium ramosum," and new isolate T4T (T = type strain), which was obtained from a marine cyanobacterial mat. "Roseococcus thiosulfatophilus" is a member of the alpha-1 subclass of the Proteobacteria and is moderately related to Rhodopila globiformis, Thiobacillus acidophilus, and Acidiphilium cryptum (level of sequence similarity, 90%). "Erythromicrobium ramosum" and isolate T4T are closely related to Erythrobacter longus and Porphyrobacter neustonensis (level of sequence similarity, 95%). These organisms are members of the alpha-4 subclass of the Proteobacteria. Strain T4T is a motile, red or orange bacterium. The major carotenoids are bacteriorubixanthinal and erythroxanthin sulfate. In vivo measurements revealed bacteriochlorophyll absorption maxima at 377, 590, 800, and 868 nm. Strain T4T grows in the presence of 5 to 96/1000 salinity and uses glucose, fructose, acetate, pyruvate, glutamate, succinate, and lactate as substrates. On the basis of its distinct phylogenetic position and phenotypic characteristics which are different from those of Erythrobacter longus, we propose that strain T4T should be placed in a new species of the genus Erythrobacter, Erythrobacter litoralis. The descriptions of "Roseococcus thiosulfatophilus" and "Erythromicrobium ramosum" are emended.

The obligate aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 contain numerous polar carotenoids. The major carotenoid of the strain RB3 was the C30 carotene-dioate (4,4′-diapocarotene-4,4′-dioate) and the respective diglycosyl ester which have never been isolated before from a bacteriochlorophyll containing bacterium. Strain E5 contains the very polar erythroxanthin sulphate. The major carotenoid bound to reaction center and light-harvesting complexes is bacteriorubixanthinal. Most of the carotenoids of both strains are not bound to the pigment-protein complexes of the photosynthetic apparatus but to the envelope fraction (cytoplasmic membrane and cell wall).

The NH2 termini of light-harvesting complex I (LHI) polypeptides alpha and beta of Rhodobacter capsulatus are thought to be involved in the assembly of the LHI complex. For a more detailed study of the role of the NH2-terminal segment of the LHI alpha protein in insertion into the intracytoplasmic membrane (ICM) of R. capsulatus, amino acids 6 to 8, 9 to 11, 12 and 13, or 14 and 15 of the LHI alpha protein were deleted. Additionally, the hydrophobic stretch of the amino acids 7 to 11 was lengthened by insertion of hydrophobic or hydrophilic amino acids. All mutations abolished the ability of the mutant strains to form a functional LHI antenna complex. All changes introduced into the LHI alpha protein strongly reduced the stability of its LHI beta partner protein in the ICM. The effects on the mutated protein itself, however, were different. Deletion of amino acids 6 to 8, 9 to 11, or 14 and 15 drastically reduced the amount of the LHI alpha protein inserted into the membrane or prevented its insertion. Deletion of amino acids 12 and 13 and lengthening of the stretch of amino acids 7 to 11 reduced the half-life of the mutated LHI alpha protein in the ICM in comparison with the wild-type LHI alpha protein. Under the selective pressure of low light, revertants which regained a functional LHI antenna complex were identified only for the mutant strain deleted of amino acids 9 to 11 of the LHI alpha polypeptide [U43 (pTPR15)]. The restoration of the LHI+ phenotype was due to an in-frame duplication of 9 bp in the pufA gene directly upstream of the site of deletion present in strain U43(pTPR15). The duplicated nucleotides code for the amino acids Lys, Ile, and Trp. Membranes purified from the revertants were different from that of the reaction center-positive LHI+ LHII- control strain U43(pTX35) in doubling of the carotenoid content and increase of the size of the photosynthetic unit. By separating the reaction center and LHI complexes of the revertants by native preparative gel electrophoresis, we confirmed that the higher amount of carotenoids was associated with the LHI proteins.

The Rhodobacter capsulatus hemA gene, coding for the enzyme delta-aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides. Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the delta-aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19. DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs. However, no resemblance was seen to the HemA protein of E. coli K12. Based on the sequence data, an ALA-requiring mutant strain of R. capsulatus was constructed by site-directed insertion mutagenesis. Introduction of a plasmid, containing the hemA gene of R. capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R. capsulatus. Transfer of the R' factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.

Reaction center (RC) and light-harvesting (LH) or antenna complexes are the major pigment-proteins of the photosynthetic apparatus of non sulfur purple bacteria. They are localized on the intracytoplasmic membranes. The LH-complexes serve to gather light-energy and funnel it to the photochemical RC where the excitation energy is transduced into a charge separation state and a redox potential difference. Reaction centers are surrounded by a constant number of core antenna complexes (8870 or B1020). Most species have a second and variable light-harvesting (LH) complex (B800–850) which interconnects the core complexes (Drews 1985). The LH complexes are oligomers of basic subunits which consist of two different small pigment-binding polypeptides α and β, having Mr of about 5000 to 7000. These polypeptides are amphiphilic proteins and span the membrane only once by a central hydrophobic domain (Fig. 1). The N- and C-terminal domains consist of polar, charged and hydrophobic amino acid residues and are exposed on the membrane surface. Two or three bacteriochlorophyll (Bchl) and one or two carotenoid molecules are bound non-covalently to the α and β polypeptides.

Facultative phototrophic bacteria such as Rhodobacter (Rb.) capsulatus can adapt to different modes of energy metabolism. Under anaerobic growth conditions light energy is captured by the antenna system of the photosynthetic apparatus and transduced into an electrochemical proton gradient across the membrane, which can be used for ATP production. In dark cultures proton motive force is generated on the expense of substrate oxidation by the respiratory chain under aerobic conditions with oxygen as electron acceptor and under anaerobic conditions with dimethyl sulfoxide, trimethylamine N-oxide, nitrate or nitrous oxide as alternative electron acceptors (Ferguson et al. 1987).

The B800–850 light harvesting complex of Rps. acidophila and the RC—B875 reaction center complex of Rps. palustris were crystallized in presence of detergents. The crystals were obtained by vapour diffusion technique using polyethylene glycol 1000 (1,2). For spectroscopic studies, the B800–850 and the RC—B875 complexes were crystallized on thin cover slides, allowing the crystals to grow to sufficient size in two dimensions. Sealed by a second cover slide, the crystals were mounted on a goniometer sample holder in a micro-spectrophotometer (3) built in our laboratory.

The photosynthetic apparatus of Rhodopseudomonas palustris contains, in addition to reaction center bacteriochlorophyll (Bchl) two spectral forms of light harvesting (LH) Bchl, i.e. LH Bchl I, characterized by an infrared absorption maximum at 880 nm (890 nm at 77 degrees K) and LH Bchl II absorbing at 805 and 855 nm (805 and 870 nm at 77 degrees K). LH Bchl I seems to be associated with a single protein species of an apparent mol. wt. of 13 000 whereas LH Bchl II is apparently associated with two proteins of mol. wts. of 9000 and 11 000. Cells in anaerobic cultures adapt to changes of light intensity 1. by variation of the size of the photosynthetic unit, i.e. the molar ratio of LH Bchl II to reaction center Bchl, 2. by variation of the number of photosynthetic units per unit of membrane area, 3. by regulation of the size of the intracytoplasmic membrane system. During adaptation of changes of oxygen partial pressure cells are able to synthesize reaction center Bchl, LH Bchl and intracytoplasmic membranes at different rates. The synthesis of reaction center Bchl and LH Bchl I are, however, coordinated with each other, while the synthesis of LH Bchl II and reaction center Bchl proceed independently.

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg ( p O 2 ). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll. Cells which were incubated at 0.5 mmHg ( p O 2 ) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of 14 C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.

Aerobically in the dark grown cultures of Rhodopseudomonas capsulata were shifted to low oxygen partial pressure for 30 min and afterwards to phototrophic conditions (anaerobic, light). During 210 min of adaptation to a phototrophic mode of life the bacteriochlorophyll (BChl) concentration increased 53-fold (doubling time 40 min) and the carotenoid content six fold. Growth was delayed. The light membrane fraction from chemotrophic and induced phototrophic cells contained low concentrations of small photosynthetic units (reaction center+light harvesting BChl B870), and low respiratory activities, especially of succinatecytochrome c oxidase. The heavy membrane fraction, i.e. the intracytoplasmic chromatophore fraction, increased during adaptation approximately 9-fold in surface area per cell, 42-fold in BChl content, 7-fold in reaction center content and 6-fold in the size of the photosynthetic unit. Phospholipid and fatty acid content and patterns changed slightly during adaptation.

Lipopolysaccharides have been isolated from eight strains of the unicellular cyanobacterium Synechococcus. Fucose, mannose, galactose, glucose and glucosamine were found in all of the lipopolysaccharides investigated. Additionally, strain-specific sugars are present and permit the chemotyping of lipopolysaccharide. Chemotype I, comprising three strains with a high G+C content of DNA (71-66 mol%), is characterized by a high rhamnose portion and by 3,6-dideoxy-d-arabino-hexose (tyvelose). Chemotype III, represented by three strains with a low G+C content of DNA (55-48 mol%), contains a mannose-polymer with small amounts of 3-O-methyl-mannose, 4-O-methyl-mannose, 2-keto-3-deoxyoctonate and mannosamine. Lipopolysaccharides of the two strains of chemotype II contain 2,3,4-tri-O-methyl-arabinose. Lipid A is difficult to split off from the polysaccharide moiety, but is present in all lipopolysaccharides from the Synechococcus strains. The presence of Lipid A is supported by the finding of β-hydroxy fatty acids, predominantly β-hydroxypalmitic acid. The distribution of branched β-hydroxy fatty acids, detected in small amounts, parallels chemotyping of lipopolysaccharide based on the sugar composition. The phosphorus content of the lipopolysaccharides is low. The pyrogenicity of lipopolysaccharides from two strains is low. Synechococcus lipopolysaccharides have little reactivity in antisera raised in rabbits against homologous cells. As far as tested they do not migrate in immunoelectrophoresis. This confirms the neutral character or low negative charge of Synechococcus lipopolysaccharides.

Strain-specific low-molecular-weight polysaccharides of different chemical compositions were obtained from cells of nine different wild-type strains of the phototrophic bacterium Rhodopseudomonas gelatinosa. The polysaccharides are free of typical capsule components like hexuronic or aminohexuronic acids but contain (except that of strain 39/2) substantial amounts of phosphorus. A number of unusual o-methyl sugars (2-o-methyl-D-galactose, 2,3-di-o-methyl-D-galactose, 2-o-methyl-L-fucose) as well as 3,6-dideoxy-D-xylo-hexose (abequose) were identified in the R. gelatinosa polysaccharides. o-Methyl and dideoxy sugars however, are typical constituents of O-specific chains of the lipopolysaccharides of gram-negative bacteria (Rhodospirillaceae and Enterobacteriaceae, respectively). Considering both the R-type character of the R. gelatinosa lipopolysaccharides and the occurrence of these strain-specific ETEROPOLYSACCHARIDES, THE ASSUMPTION SEEMS TO BE JUSTIFIED THAT THE LOW-MOLECULAR-WEIGHT POLYSACCHARIDES ARE RELATED TO O-specific chains of lipopolysaccharides (haptens) rather than to capsular or slime antigens. In serological terms the polysaccharides of R. gelatinosa have to be classified as K-antigens. They are able to cover the O-specificity of the respective different strains and confer on them additional specificity which is demonstrable by bacterial agglutination.

By means of sucrose density centrifugation three membrane fractions, named “light, medium and heavy” have been isolated from cells of Rhodopseudomonas capsulata strain 37b4, adapting from chemotrophic to phototrophic growth conditions. Succinate dehydrogenase activity of aerobically grown cells was mainly confined to the heavy (chromatophore) fraction. Upon changing to phototrophic conditions the activity of the succinate dehydrogenase increased in the medium and light fraction. All fractions contain bacteriochlorophyll. NADH dehydrogenase of chemotrophically grown cells was enriched in the light and medium fraction but is increased in the heavy fraction under phototrophic growth conditions. The capacity of photophosphorylation is high in the light and heavy fraction. The results indicate a differentially incorporation of functional subunits into specific parts of the membrane system during membrane differentiation.