Whereas most nontyphoidal Salmonella (NTS) are associated with gastroenteritis, there has been a dramatic increase in reports of NTS-associated invasive disease in sub-Saharan Africa. Salmonella enterica serovar Typhimurium isolates are responsible for a significant proportion of the reported invasive NTS in this region. Multilocus sequence analysis of invasive S. Typhimurium from Malawi and Kenya identified a dominant type, designated ST313, which currently is rarely reported outside of Africa. Whole-genome sequencing of a multiple drug resistant (MDR) ST313 NTS isolate, D23580, identified a distinct prophage repertoire and a composite genetic element encoding MDR genes located on a virulence-associated plasmid. Further, there was evidence of genome degradation, including pseudogene formation and chromosomal deletions, when compared with other S. Typhimurium genome sequences. Some of this genome degradation involved genes previously implicated in virulence of S. Typhimurium or genes for which the orthologs in S. Typhi are either pseudogenes or are absent. Genome analysis of other epidemic ST313 isolates from Malawi and Kenya provided evidence for microevolution and clonal replacement in the field.

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The centromeric DNA (CEN3) from yeast chromosome III has been isolated on a 1.6 kilobase-pair segment of DNA located near the centromere-linked CDC10 locus of Saccharomyces cerevisiae. When present on a plasmid carrying a yeast chromosomal replicator, CEN3 enables that plasmid to function as a chromosome both mitotically and meiotically. Minichromosomes containing CEN3 are stable in mitosis and segregate as ordinary yeast chromosomes in the first and second meiotic divisions.

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.

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In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (⌿) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known ⌿ synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative ⌿ synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative ⌿ synthase domain of Cbf5p. Yeast strains expressing these mutated cbf5 genes in a cbf5⌬ null background are viable at 25°C but display pronounced cold-and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of ⌿ in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of ⌿ in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks ⌿ in rRNA. A subset of mutations in the ⌿ synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the ⌿ synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.In eukaryotes the biosynthesis of rRNA occurs in a specialized organelle known as the nucleolus (33,41,46,56). rRNA is transcribed by RNA polymerase I (Pol I) as a single large precursor, which undergoes a series of endo-and exonucleolytic cleavages to produce mature rRNA species. In the yeast Saccharomyces cerevisiae, the 35S pre-rRNA precursor is processed to produce mature 18S, 5.8S, and 25S RNAs (54). The 5S rRNA and ribosomal proteins are imported into the nucleolus for assembly into precursors of the 40S and 60S ribosomal subunits before their export to the cytoplasm (16,41,46). An interesting feature of rRNA maturation is the extensive modification the 35S precursor undergoes prior to subsequent cleavage events (29,40,39). One such modification, isomerization of uridine to pseudouridine (⌿), is by far the most abundant posttranscriptional modification of rRNA (29,40,39). Formation of ⌿ is also known to occur in tRNAs (49), small nuclear RNAs (sn...

Here we discuss the evolution of the northern Australian Staphylococcus aureus isolate MSHR1132 genome. MSHR1132 belongs to the divergent clonal complex 75 lineage. The average nucleotide divergence between orthologous genes in MSHR1132 and typical S. aureus is approximately sevenfold greater than the maximum divergence observed in this species to date. MSHR1132 has a small accessory genome, which includes the well-characterized genomic islands, νSAα and νSaβ, suggesting that these elements were acquired well before the expansion of the typical S. aureus population. Other mobile elements show mosaic structure (the prophage φSa3) or evidence of recent acquisition from a typical S. aureus lineage (SCCmec, ICE6013 and plasmid pMSHR1132). There are two differences in gene repertoire compared with typical S. aureus that may be significant clues as to the genetic basis underlying the successful emergence of S. aureus as a pathogen. First, MSHR1132 lacks the genes for production of staphyloxanthin, the carotenoid pigment that confers upon S. aureus its characteristic golden color and protects against oxidative stress. The lack of pigment was demonstrated in 126 of 126 CC75 isolates. Second, a mobile clustered regularly interspaced short palindromic repeat (CRISPR) element is inserted into orfX of MSHR1132. Although common in other staphylococcal species, these elements are very rare within S. aureus and may impact accessory genome acquisition. The CRISPR spacer sequences reveal a history of attempted invasion by known S. aureus mobile elements. There is a case for the creation of a new taxon to accommodate this and related isolates.

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome H behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes. (8,9). In contrast, however, the centromere regions of the fission yeast Schizosaccharomyces pombe resemble those of higher eucaryotes in containing several classes of repeated DNA sequences which encompass many kilobases (kb) of DNA on each of the three S. pombe chromosomes (6,7,10,24). Centromeric repeats designated K, L, and B are heterochromatic-like and untranscribed (10). The centromere regions of the three S. pombe chromosomes, including all the centromere-specific DNA sequence repeats, reside on three large genomic SalI restriction fragments of 65, 100, and 150 kb, derived from chromosomes I, II, and III, respectively (10).Recently Gutz et al. (12). DNA transformations of S. pombe were performed as described before (16). Stable Ura+ transformants obtained by site-directed integration of pSp500 (Fig. 1) into SBPD400 were identified as follows. A total of 240 Ura+ transformant colonies were picked onto nonselective medium and subsequently replica-plated for seven rounds of nonselective growth before they were replica-plated to medium selecting for the Ura+ phenotype. Those clones (14 total) that were still Ura+ after several rounds of nonselective growth and that contained sequences hybridizing to pBR322 were characterized further.