Disruption of a gene for an alternative sigma factor, designated sigF, in the freshwater, unicellular cyanobacterium Synechocystis sp, strain PCC6803 resulted in a pleiotropic phenotype. Most notably, this mutant lost phototactic movement with a concomitant loss of pill, which are abundant on the surface of wild-type cells. The sigF mutant also secreted both high levels of yellow-brown and UV. absorbing pigments and a polypeptide that is similar to a large family of extracellular proteins that includes the hemolysins. Furthermore, the sigF mutant had a dramatically reduced level of the transcript from two tandemly arranged bill genes (sll1694 and sll1695), which encode major structural components of type IV pill, Inactivation of these piL-1 genes eliminated phototactic movement, though some pill were still present in this strain. Together, these results demonstrate that SigF plays a critical role in motility via the control of pill formation and is also likely to regulate other features of the cell surface. Furthermore, the data provide evidence that type IV pill are required for phototactic movement in certain cyanobacteria and suggest that different populations of pill present on the Synechocystis cell surface may perform different functions.

We have recently shown that phototactic movement in the unicellular cyanobacterium Synechocystis sp. PCC6803 requires type IV pilins. To elucidate further type IV pilus-dependent motility, we inactivated key genes implicated in pilus biogenesis and function. Wild-type Synechocystis sp. PCC6803 cells have two morphologically distinct pilus types (thick and thin pili). Of these, the thick pilus morphotype, absent in a mutant disrupted for the pilin-encoding pilA1 gene, appears to be required for motility. The thin pilus morphotype does not appear to be altered in the pilA1 mutant, raising the possibility that thin pili have a function distinct from that of motility. Mutants disrupted for pilA2, which encodes a second pilin-like protein, are still motile and exhibit no difference in morphology or density of cell-surface pili. In contrast, inactivation of pilD (encoding the leader peptidase) or pilC (encoding a protein required for pilus assembly) abolishes cell motility, and both pilus morphotypes are absent. Thus, the PilA1 polypeptide is required for the biogenesis of the thick pilus morphotype which, in turn, is necessary for motility (hence we refer to them as type IV pili). Furthermore, PilA2 is critical neither for motility nor for pilus biogenesis. Two genes encoding proteins with similarity to PilT, which is considered to be a component of the motor essential for type IV pilus-dependent movement, were also inactivated. A pilT1 mutant is (i) non-motile, (ii) hyperpiliated and (iii) accumulates higher than normal levels of the pilA1 transcript. In contrast, pilT2 mutants are motile, but are negatively phototactic under conditions in which wild-type cells are positively phototactic.

Global identification of differentially regulated genes in prokaryotes is constrained because the mRNA does not have a 3' polyadenylation extension; this precludes specific separation of mRNA from rRNA and tRNA and synthesis of cDNAs from the entire mRNA population, from ledge of the entire genome sequence of Synechocystis sp. strain PCC 6803 has enabled us to develop a differential display procedure that takes advantage of a short palindromic sequence that is dispersed throughout the Synechocystis sp. strain PCC 6803 genome. This sequence, designated the HIP (highly iterated palindrome) element, occurs in approximately half of the Synechocystis sp, strain PCC 6803 genes but is absent in rRNA and tRNA. genes. To determine the feasibility of exploiting the HIP element, alone or in combination with specific primer subsets, for analyzing differential gene expression, we used HIP-based primers to identify light intensity-regulated genes. Several gene fragments, including those encoding ribosomal proteins and phycobiliprotein subunits, were differentially amplified from RNA templates derived from cells grown in low light or exposed to high light for 3 h. One novel finding was that expression of certain genes of the pho regulon, which are under the control of environmental phosphate levels, were markedly elevated in high light. High-light activation of pho regulon genes correlated with elevated growth rates that occur when the cells are transferred from low to high fight. These results suggest that in high light, the rate of growth of Synechocystis sp. strain PCC 6803 exceeds its capacity to assimilate phosphate, which, in turn, may trigger a phosphate starvation response and activation of the pho regulon.

Concentrating algal cells by flocculation as a prelude to centrifugation could significantly reduce the energy and cost of harvesting the algae. However, how variation in phenotypic traits such as cell surface features, cell size and motility alter the efficiency of metal cation and pH-induced flocculation is not well understood. Our results demonstrate that both wild-type and cell wall-deficient strains of the green unicellular alga Chlamydomonas reinhardtii efficiently flocculate (> 90%) at an elevated pH of the medium (pH 11) upon the addition of divalent cations such as calcium and magnesium (> 5 mM). The trivalent ferric cation (at 10 mM) proved to be essential for promoting flocculation under weak alkaline conditions (pH similar to 8.5), with a maximum efficiency that exceeded 95 and 85% for wild-type CC1690 and the cell wall-deficient sta6 mutant, respectively. Near complete flocculation could be achieved using a combination of 5 mM calcium and a pH > 11, while the medium recovered following cell removal could be re-cycled without affecting algal growth rates. Moreover, the absence of starch in the cell had little overall impact on flocculation efficiency. These findings contribute to our understanding of flocculation in different Chlamydomonas strains and have implications with respect to inexpensive methods for harvesting algae with different phenotypic traits. Additional research on the conditions (e.g., pH and metal ions) used for efficient flocculation of diverse algal groups with diverse characteristics, at both small and large scale, will help establish inexpensive procedures for harvesting cell biomass.

Arabidopsis thaliana, a small annual plant belonging to the mustard family, is the subject of study by an estimated 7000 researchers around the world. In addition to the large body of genetic, physiological and biochemical data gathered for this plant, it will be the first higher plant genome to be completely sequenced, with completion expected at the end of the year 2000. The sequencing effort has been coordinated by an international collaboration, the Arabidopsis Genome initiative (AGI). The rationale for intensive investigation of Arabidopsis is that it is an excellent model for higher plants. in order to maximize use of the knowledge gained about this plant, there is a need for a comprehensive database and information retrieval and analysis system that will provide user-friendly access to Arabidopsis information. This paper describes the initial steps we have taken toward realizing these goals in a project called The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org).

In land plants, linear tetrapyrrole (bilin)-based phytochrome photosensors optimize photosynthetic light capture by mediating massive reprogramming of gene expression. But, surprisingly, many green algal genomes lack phytochrome genes. Studies of the heme oxygenase mutant (hmox1) of the green alga Chlamydomonas reinhardtii suggest that bilin biosynthesis in plastids is essential for proper regulation of a nuclear gene network implicated in oxygen detoxification during dark-to-light transitions. hmox1 cannot grow photoautotrophically and photoacclimates poorly to increased illumination. We show that these phenotypes are due to reduced accumulation of photosystem I (PSI) reaction centers, the PSI electron acceptors 5'-monohydroxyphylloquinone and phylloquinone, and the loss of PSI and photosystem II antennae complexes during photoacclimation. The hmox1 mutant resembles chlorophyll biosynthesis mutants phenotypically, but can be rescued by exogenous biliverdin IX alpha, the bilin produced by HMOX1. This rescue is independent of photosynthesis and is strongly dependent on blue light. RNA-seq comparisons of hmox1, genetically complemented hmox1, and chemically rescued hmox1 reveal that tetrapyrrole biosynthesis and known photoreceptor and photosynthesis-related genes are not impacted in the hmox1 mutant at the transcript level. We propose that a bilin-based, blue-light-sensing system within plastids evolved together with a bilin-based retrograde signaling pathway to ensure that a robust photosynthetic apparatus is sustained in light-grown Chlamydomonas.

Comparisons of codon frequencies of genes to several gene classes are used to characterize highly expressed and alien genes on the Synechocystis PCC6803 genome, The primary gene classes include the ensemble of all genes (average gene), ribosomal protein (RP) genes, translation processing factors (TF) and genes encoding chaperone/degradation proteins (CH), A gene is predicted highly expressed (PHX) if its codon usage is close to that of the RP/TF/CH standards but strongly deviant from the average gene. Putative alien (PA) genes are those for which codon usage is significantly different from all four classes of gene standards. In Synechocystis, 380 genes were identified as PHX, The genes with the highest predicted expression levels include many that encode proteins vital for photosynthesis. Nearly all of the genes of the RP/TF/CH gene classes are PHX, The principal glycolysis enzymes, which may also function in CO2 fixation, are PHX, while none of the genes encoding TCA cycle enzymes are PHX, The PA genes are mostly of unknown function or encode transposases, Several PA genes encode polypeptides that function in lipopolysaccharide biosynthesis. Both PHX and PA genes often form significant clusters (operons), The proteins encoded by PHX and PA genes are described with respect to functional classifications, their organization in the genome and their stoichiometry in multi-subunit complexes.

Six members of a multigene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex from female gametophytes of the brown alga Macrocystis pyrifera have been cloned and characterized. The deduced amino acid sequences are very similar to those of fucoxanthin chlorophyll binding proteins (Fcp) from the diatom Phaeodactylum tricornutum and exhibit limited homology to chlorophyll a/b binding (Cab) polypeptides from higher plants. The primary translation products from the M. pyrifera fcp genes are synthesized as higher molecular weight precursors that are processed prior to their assembly into the Fcp complex. The presumed N-terminal 40-amino acid presequence of the Fcp precursor polypeptide has features resembling that of a signal sequence. This presequence may be required for the protein to transverse the endoplasmic reticulum that surrounds the plastid in brown algae. A subsequent targeting step would be required for the protein to cross the double membrane of the plastid envelope. M. pyrifera fcp transcripts are of two sizes, 1.2 and 1.6 kb. The size difference is accounted for by the length of the 3' untranslated region, which can be up to 1000 bases. Transcript abundance's of members of the fcp gene family are dependent on light quantity, light quality, or both. Transcript levels of one gene increased approximately five- to tenfold in thalli grown in low intensity relative to high intensity white or blue light. Transcripts from this gene also significantly increase in red light relative to blue light at equivalent light intensities.