The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

The chloroplast genome of Pelargonium x hortorum has been completely sequenced. It maps as a circular molecule of 217,942 bp and is both the largest and most rearranged land plant chloroplast genome yet sequenced. It features 2 copies of a greatly expanded inverted repeat (IR) of 75,741 bp each and, consequently, diminished single-copy regions of 59,710 and 6,750 bp. Despite the increase in size and complexity of the genome, the gene content is similar to that of other angiosperms, with the exceptions of a large number of pseudogenes, the recognition of 2 open reading frames (ORF56 and ORF42) in the trnA intron with similarities to previously identified mitochondrial products (ACRS and pvs-trnA), the losses of accD and trnT-ggu and, in particular, the presence of a highly divergent set of rpoA-like ORFs rather than a single, easily recognized gene for rpoA. The 3-fold expansion of the IR (relative to most angiosperms) accounts for most of the size increase of the genome, but an additional 10% of the size increase is related to the large number of repeats found. The Pelargonium genome contains 35 times as many 31 bp or larger repeats than the unrearranged genome of Spinacia. Most of these repeats occur near the rearrangement hotspots, and 2 different associations of repeats are localized in these regions. These associations are characterized by full or partial duplications of several genes, most of which appear to be nonfunctional copies or pseudogenes. These duplications may also be linked to the disruption of at least 1 but possibly 2 or 3 operons. We propose simple models that account for the major rearrangements with a minimum of 8 IR boundary changes and 12 inversions in addition to several insertions of duplicated sequence.

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA , which codes for translation initiation factor 1, in Ͼ 300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in ‫ف‬ 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants. INTRODUCTIONMany genes have been lost from the chloroplast genome during plant and algal evolution. Most of these losses occurred in the murky interval between the original endosymbiosis of a cyanobacterium (with perhaps 2000 proteincoding genes) and the last common ancestor of all existing chloroplast genomes (with ‫ف‬ 210 protein-coding genes; . Many other genes were lost during the early evolution of photosynthetic eukaryotes, often in parallel in different algal lineages, and some of these losses were the result of gene transfers to the nuclear genome . During the evolution of land plants, relatively few changes occurred to the set of genes found in chloroplast DNA (cpDNA) Palmer and Delwiche, 1998). Nonetheless, the most recent changes are likely to provide the most information about the evolutionary mechanisms involved.Among the six completely sequenced chloroplast genomes from angiosperms (excluding the nonphotosynthetic plant Epifagus virginiana ; Wolfe et al., 1992a), 74 proteincoding genes are held in common and an additional five are present in only some species. These five genes are accD , ycf1 , and ycf2 (pseudogenes in rice and maize; Hiratsuka et al., 1989;Maier et al., 1995), rpl23 (pseudogene in spinach; Thomas et al., 1988), and infA (pseudogene in tobacco, Arabidopsis, and Oenothera elata ; Shinozaki et al., 1986;Wolfe et al., 1992b;Sato et al., 1999; Hupfer et al., 2000). Other chloroplast gene losses in angiosperms that have been confirmed by sequencing include rpl22 , rps16 , and ycf4 (open reading frame 184), all of which have been lost in 1 To whom correspondence should be addressed. E-mail (in Dublin) khwolfe@tcd.ie; fax 353-1-6798558. 646The Plant Cell some or all legumes (Gantt et al., 1991;Nagano et al., 1991; Doyle et al., 1995; K.H. Wolfe, unpublished data), and ycf2 and ndhF , both of which have been lost ...

Most chloroplast and mitochondrial proteins are encoded by nuclear genes that once resided in the organellar genomes. Transfer of most of these genes appears to have occurred soon after the endosymbiotic origin of organelles, and so little is known about the process. Our efforts to understand how chloroplast genes are functionally transferred to the nuclear genome have led us to discover the most recent evolutionary gene transfer yet described. The gene rpl22, encoding chloroplast ribosomal protein CL22, is present in the chloroplast genome of all plants examined except legumes, while a functional copy of rpl22 is located in the nucleus of the legume pea. The nuclear rpl22 gene has acquired two additional domains relative to its chloroplast ancestor: an exon encoding a putative N‐terminal transit peptide, followed by an intron which separates this first exon from the evolutionarily conserved, chloroplast‐derived portion of the gene. This gene structure suggests that the transferred region may have acquired its transit peptide by a form of exon shuffling. Surprisingly, phylogenetic analysis shows that rpl22 was transferred to the nucleus in a common ancestor of all flowering plants, at least 100 million years preceding its loss from the legume chloroplast lineage.

We reexamined the influence of fimE, also referred to as hyp, on type 1 fimbriation in Escherichia coli K-12. We found that one strain used previously and extensively in the analysis of type 1 fimbriation, strain CSH50, is in fact a fimE mutant; the fimE gene of CSH50 contains a copy of the insertion sequence IS1. Using a recently described allelic exchange procedure, we transferred the fimE::IS1 allele from CSH50 to our present wild-type strain, MG1655. Characterization of this IS1-containing strain (AAEC137), together with another fimE mutant of MG1655 (AAEC143), led to two conclusions about the role of fimE. First, the formation of phase variant colony types, reported widely in strains of E. coli, depends on mutation of fimE, at least in K-12 strain MG1655. Here we showed that this phenomenon reflects the ability of fimE to stimulate the rapid inversion of the fim invertible element from on to off when the bacteria are grown on agar. Second, our analysis of fimE mutants, which is limited to chromosomal constructs, provided no evidence that they are hyperfimbriate. We believe that these results, which are at odds with a previous study using fim-containing multicopy plasmids, reflect differences in gene copy number.

Epichloae (Epichloë and Neotyphodium species; Clavicipitaceae) are fungi that live in systemic symbioses with cool-season grasses, and many produce alkaloids that are deterrent or toxic to herbivores. The epichloae colonize much of the aerial plant tissues, and most benignly colonize host seeds to transmit vertically. Of their four chemical classes of alkaloids, the ergot alkaloids and indole-diterpenes are active against mammals and insects, whereas peramine and lolines specifically affect insects. Comparative genomic analysis of Clavicipitaceae reveals a distinctive feature of the epichloae, namely, large repeat blocks in their alkaloid biosynthesis gene loci. Such repeat blocks can facilitate gene losses, mutations, and duplications, thus enhancing diversity of alkaloid structures within each class. We suggest that alkaloid diversification is selected especially in the vertically transmissible epichloae.

The phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a short DNA element. This element (switch) acts in cis to control transcription offimA, the major fimbrial subunit gene. Thus,fimA4 is transcribed when the switch is in one orientation (the on orientation) but not the other (the off orientation). Thefim inversion requires eitherfimB (on-to-off or off-to-on inversion) orfimE (on-to-offinversion only), as well as integration host factor, and is also influenced by the abundant DNA-binding protein H-NS. Here we report that an additional gene, lIrp, a factor known to influence the expression of both Pap and K99 fimbriae, is also required for normal activity of the fim switch. The frequencies of both fimB-promoted and fimE-promoted inversions, and consequently the phase variation of type 1 fimbriation, are lower in lIrp mutants. Lrp affects slightly the transcription of both fimB (which is increased) and fimE (which is decreased). We believe that these alterations in fimB and fimE transcription alone are unlikely to account for the sharp reduction in switching found in lrp mutants.Fimbriae, proteinaceous appendages produced by many gram-negative bacteria, promote adherence between bacterial and host cells. Although attachment is probably an important step in pathogenesis, fimbriae are also excellent immunogens (13,30,49,50), and their expression presumably leaves the cell vulnerable to the host's immune defenses. Perhaps to help avoid the immune system, the expression of many fimbrial types is phase variable. Thus, the ability to undergo phase variation, using a range of mechanisms (reviewed in reference 53) that involve changes in DNA methylation (8) and DNA inversion (1, 35), as well as other DNA rearrangements (24,38,60), may in itself be a pathogenicity factor (15).The phase variation of type 1 fimbriation in Escherichia coli involves inversion of a 314-bp DNA element (1). This element (switch), which is situated immediately upstream of fimA, acts in cis to control the transcription of fimA, the major fimbrial subunit gene (27,43

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA , which codes for translation initiation factor 1, in 300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.