Assessment of ciliates in the sheep rumen by DGGE.

AIMS: This work was carried out to develop a rapid molecular profiling technique to screen ciliate populations in the rumen of sheep. METHODS AND RESULTS: DGGE was used to study the ciliate diversity in the rumen of sheep. There was considerable variation between sheep which were co-housed, and fed the same diet. However, no difference in the major banding patterns was detected, when samples were collected from a single sheep sampled at different points. Following dietary changes, use of a pair-wise comparison of lanes, demonstrated that although there was still diversity between the ciliate population of sheep, the effects as a result of dietary changes were greater. CONCLUSIONS: The technique generated molecular profiles which are sufficiently different to allow comparison between samples, and to permit molecular ecological studies on the rumen ciliate population. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this study means that ciliate diversity in the rumen may now be studied by those unfamiliar with morphological identification of these organisms.

Expression and phylogeny of the multiple antenna genes of the low-light-adapted strain Prochlorococcus marinus SS120 (Oxyphotobacteria).

In contrast to typical cyanobacteria, Prochlorococcus strains possess an intrinsic divinyl-chlorophyll (Chl) a/b-protein complex instead of phycobilisomes as the major light-harvesting system. These pigment-protein complexes are encoded by a variable number of pcb genes depending on the ecotype to which the Prochlorococcus strain belongs: low-light-adapted strains possess several pcb gene copies whereas only a single copy is present in high-light-adapted strains. In this study, the light-regulated expression of the seven pcb genes of Prochlorococcus marinus SS120 was examined. The pcbF gene was found to exhibit a high turnover and its mRNA could only be detected as a degraded product under all light conditions. Steady-state levels of transcripts originating from the six other pcb gene copies varied over several orders of magnitude but were not significantly differentially regulated by light intensity. Transcript levels of most pcb genes increased between 4.5 and 8.5 micromol quanta m(-2) s(-1), peaked at 45 micromol m(-2) s(-1) and decreased at the highest irradiance (72 micromol m(-2) s(-1)). A phylogenetic analysis of the Pcb proteins and other members of the six-helix Chl protein superfamily revealed that PcbC and PcbG make a separate cluster with regard to the other Pcbs from SS120. In contrast, Pcb sequences from four high-light-adapted Prochlorococcus sp. strains were found to cluster together and to be less variable than SS120 Pcbs. Thus, pcb genes likely evolved at a different rate in the two Prochlorococcus ecotypes. Their early multiplication and diversification is likely a key factor in the successful adaptation of some genotypes to very-low-light conditions.

Multiplication of antenna genes as a major adaptation to low light in a marine prokaryote.

Two ecotypes of the prokaryote Prochlorococcus adapted to distinct light niches in the ocean have been described recently. These ecotypes are characterized by their different (divinyl-) chlorophyll (Chl) a to Chl b ratios and 16S rRNA gene signatures, as well as by their significantly distinct irradiance optima for growth and photosynthesis [Moore, L. R., Rocap, G. & Chisholm, S. W. (1998) Nature (London) 393, 464-467]. However, the molecular basis of their physiological differences remained, so far, unexplained. In this paper, we show that the low-light-adapted Prochlorococcus strain SS120 possesses a gene family of seven transcribed genes encoding different Chl a/b-binding proteins (Pcbs). In contrast, Prochlorococcus sp. MED4, a high-light-adapted ecotype, possesses a single pcb gene. The presence of multiple antenna genes in another low-light ecotype (NATL2a), but not in another high-light ecotype (TAK9803-2), is demonstrated. Thus, the multiplication of pcb genes appears as a key factor in the capacity of deep Prochlorococcus populations to survive at extremely low photon fluxes.

Rapid evolutionary divergence of Photosystem I core subunits PsaA and PsaB in the marine prokaryote Prochlorococcus.

The nucleotide sequences of the genes coding for the subunits of the Photosystem I (PS I) core, PsaA and PsaB were determined for the marine prokaryotic oxyphototrophs Prochlorococcus sp. MED4 (CCMP1378), P. marinus SS120 (CCMP1375) and Synechococcus sp. WH7803. Divergence of these sequences from those of both freshwater cyanobacteria and higher plants was remarkably high, given the conserved nature of PsaA and PsaB proteins. In particular, the PsaA of marine prokaryotes showed several specific insertions and deletions with regard to known PsaA sequences. Even in between the two Prochlorococcus strains, which correspond to two genetically different ecotypes with shifted growth irradiance optima, the sequence identity was only 80.2% for PsaA and 88.9% for PsaB. Possible causes and implications of the fast evolution rates of these two PS I core subunits are discussed.

The 38 kDa chlorophyll a/b protein of the prokaryote Prochlorothrix hollandica is encoded by a divergent pcb gene.

The chlorophyll (Chl) a/b proteins of the photosynthetic prokaryotes appear to have evolved by gene duplication and divergence of the core Chl a antenna family, which also includes CP43 and CP47 and the iron-stress induced Chl a-binding IsiA proteins. We show here that Prochlorothrix hollandica has a cluster of three pcb (prochlorophyte chlorophyll b) genes which are co-transcribed. The major antenna polypeptides of 32 and 38 kDa are encoded by pcbA and pcbC respectively. The pcbC gene is significantly divergent from the other two and may have originated by a gene duplication independent of the one that led to isiA and the other prochlorophyte pcb genes. The distant relatedness of the three prochlorophyte genera implies that not only the ability to make Chl b and use it for light-harvesting arose independently in the three lineages, but also that the pcb genes may have arisen as the result of independent gene duplications in each lineage.

Independent evolution of the prochlorophyte and green plant chlorophyll a/b light-harvesting proteins.

The prochlorophytes are oxygenic prokaryotes differing from other cyanobacteria by the presence of a light-harvesting system containing both chlorophylls (Chls) a and b and by the absence of phycobilins. We demonstrate here that the Chl a/b binding proteins from all three known prochlorophyte genera are closely related to IsiA, a cyanobacterial Chl a-binding protein induced by iron starvation, and to CP43, a constitutively expressed Chl a antenna protein of photosystem II. The prochlorophyte Chl a/b protein (pcb) genes do not belong to the extended gene family encoding eukaryotic Chl a/b and Chl a/c light-harvesting proteins. Although higher plants and prochlorophytes share common pigment complements, their light-harvesting systems have evolved independently.

Coexistence of phycoerythrin and a chlorophyll a/b antenna in a marine prokaryote.

Prochlorococcus marinus CCMP 1375, a ubiquitous and ecologically important marine prochlorophyte, was bound to possess functional genes coding for the alpha and beta subunits of a phycobiliprotein. The latter is similar to phycoerythrins (PE) from marine Synechococcus cyanobacteria and bind a phycourobilin-like pigment as the major chromophore. However, differences in the sequences of the alpha and beta chains compared with known PE subunits and the presence of a single bilin attachment site on the alpha subunit designate it as a novel PE type, which we propose naming PE-III. P. marinus is the sole prokaryotic organisms known so far that contains chlorophylls a and b as well as phycobilins. These data strongly suggest that the common ancestor of prochlorophytes and the Synechococcus cyanobacteria contained phycobilins. Flow cytometric data from the tropical Pacific Ocean provide evidence that deep populations of Prochlorococcus possess low amounts of a PE-like pigment, which could serve either in light harvesting or nitrogen storage or both.

Biochemical characterization of protein composition and protein phosphorylation patterns in stacked and unstacked thylakoid membranes of the prochlorophyte Prochlorothrix hollandica.

We describe a procedure to separate stacked and unstacked membranes from the prochlorophyte Prochlorothrix hollandica that is based on methods used for the separation of grana and stroma thylakoids from chloroplasts. Stacked membranes were isolated from Triton X-100-treated whole thylakoid preparations, unstacked membranes from French press disrupted cells. Membrane fractions were isolated by differential centrifugation. The stacked membranes were enriched in photosystem (PS) II and a chlorophyll a/b-binding antenna complex, whereas the unstacked membranes contained PS I and the ATP synthase. No evidence for a PS I-associated chlorophyll a/b antenna system was obtained. The PS II-associated chlorophyll a/b antenna complex is composed of several apoproteins in the molecular mass range from 32 to 38 kDa. The 38-kDa protein of this complex becomes phosphorylated on its stromal surface by a light-activated kinase, but, unlike the light-harvesting II complex of chloroplasts, it does not migrate from grana to stroma membrane regions. Overall, the thylakoid membranes of Prochlorothrix exhibit a remarkably similar organization to those of chloroplasts, especially in terms of the non-random distribution of protein complexes between grana and stroma thylakoid membranes. However, in contrast to the rapidly reversible phosphorylation-dependent state 1-state 2 response of light-harvesting complex associated with PS II in chloroplasts, the slowly reversible phosphorylation in Prochlorothrix leads only to a functional uncoupling from PS II but not to its redistribution between stacked and unstacked membrane regions.