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Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Most chloroplastic proteins are cytosolically synthesized and posttranslationally transported to their proper locations.' Two examples of this group of proteins are ferredoxin and plastocyanin, both of which are metal-containing components of the photosynthetic electron-transport chain.The import process for these two proteins includes the insertion of the metal ions to produce the holo forms of the proteins. We show here that in vitro translated precursor. proteins of ferredoxin and plastocyanm are synthesized as apo forms and are assembled into their respective holo forms after being imported into isolated chloroplasts. We also provide evidence that only mature-sized proteins are compete'nt to be assembled into holo forms.Most chloroplastic proteins are encoded in the nucleus and synthesized in the cytosol as higher molecular weight precursors with amino-terminal-extensions called transit peptides. They are imported into chloroplasts' posttranslationally. The import process is usually divided into the following steps: (i) binding of precursors to the outer envelope membrane; (ii) translocation of polypeptides across the outer and inner envelope membranes; (iii) removal of transit peptides by the stromal processing protease; and (iv) depending on the protein, either further sorting into other chloroplastic compartments and/or assembly into their respective complexes (1). Most import studies have been performed using an in vitro reconstituted system in which radiolabeled precursors are synthesized by in vitro transcription and translation from cloned genes and are subsequently imported into isolated chloroplasts.Precursors to ferredoxin (prFD) and plastocyanin (prPC) are two proteins that have been examined in transport studies (2). The mature forms of these proteins are metalloproteins that function in photosynthetic electron transport. One unsolved aspect of their biogenesis concerns assembly of their metal-ion prosthetic groups. Although these two proteins follow the general steps of import described above,' current evidence is inadequate to determine when during the import process prosthetic groups are added. An understanding of when the metal-ion centers are assembled will have important implications for possible import mechanisms. For example, if the metal-ion centers are added in the cytosol, the precursors would then have to carry the metal-ion centers across the envelope during import. If the metal-ion centers are assembled inside chloroplasts, this raises questions about whether assembly occurs before or after proteolytic processing. Imported precursors and mature-sized proteins may have different conformations and only one ofthem may be competent for prosthetic group assembly.Higher plant ferredoxin (FD) contains a 2Fe-2S-type ironsulfur center and functions exclusively in the stroma of chloroplasts. A pathway for introducing the iron-sulfur center into spinach FD in isolated chloroplasts has been described (3). Isolated chloroplasts were incubated with exogenous cysteine and the sulfur atoms ...

Whole-genome sequencing, particularly in fungi, has progressed at a tremendous rate. More difficult, however, is experimental testing of the inferences about gene function that can be drawn from comparative sequence analysis alone. We present a genome-wide functional characterization of a sequenced but experimentally understudied budding yeast, Saccharomyces bayanus var. uvarum (henceforth referred to as S. bayanus), allowing us to map changes over the 20 million years that separate this organism from S. cerevisiae. We first created a suite of genetic tools to facilitate work in S. bayanus. Next, we measured the gene-expression response of S. bayanus to a diverse set of perturbations optimized using a computational approach to cover a diverse array of functionally relevant biological responses. The resulting data set reveals that gene-expression patterns are largely conserved, but significant changes may exist in regulatory networks such as carbohydrate utilization and meiosis. In addition to regulatory changes, our approach identified gene functions that have diverged. The functions of genes in core pathways are highly conserved, but we observed many changes in which genes are involved in osmotic stress, peroxisome biogenesis, and autophagy. A surprising number of genes specific to S. bayanus respond to oxidative stress, suggesting the organism may have evolved under different selection pressures than S. cerevisiae. This work expands the scope of genome-scale evolutionary studies from sequence-based analysis to rapid experimental characterization and could be adopted for functional mapping in any lineage of interest. Furthermore, our detailed characterization of S. bayanus provides a valuable resource for comparative functional genomics studies in yeast.

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