Search

We analyzed the within-household evolution of two household-associated Escherichia coli strains from pandemic clonal group ST131- H 30, using isolates recovered from five individuals within two families, each of which had a distinct strain. Family 1's strain was represented by a urine isolate from the index patient (older sister) with recurrent cystitis and a blood isolate from her younger sister with fatal urosepsis. Family 2's strain was represented by a urine isolate from the index patient (father) with pyelonephritis and renal abscesses, blood and kidney drainage isolates from the daughter with emphysematous pyelonephritis, and urine and fecal isolates from the mother with cystitis. Collectively, the several variants of each family's strain had accumulated a total of 8 (family 1) and 39 (family 2) point mutations; no two isolates were identical. Of the 47 total mutations, 36 resulted in amino acid changes or truncation of coded proteins. Fourteen such mutations (39%) targeted genes encoding transcriptional regulators, and 9 (25%) involved DNA-binding transcription factors (TFs), which significantly exceeded the relative contribution of TF genes to the isolates' genomes (∼6%). At least one-half of the transcriptional regulator mutations were inactivating, based on phenotypic and/or transcriptional analysis. In particular, inactivating mutations in the global regulator LrhA (repressor of type 1 fimbriae and flagella) occurred in the blood isolates from both households and increased the virulence of E. coli strains in a murine sepsis model. The results indicate that E. coli undergoes adaptive evolution between and/or within hosts, generating subpopulations with distinctive phenotypes and virulence potential. IMPORTANCE The clonal evolution of bacterial strains associated with interhost transmission is poorly understood. We characterized the genome sequences of clonal descendants of two Escherichia coli strains, recovered at different time points from multiple individuals within two households who had different types of urinary tract infection. We found evidence that the E. coli strains underwent extensive mutational diversification between and within these individuals, driven disproportionately by inactivation of transcriptional regulators. In urosepsis isolates, the mutations observed in the global regulator LrhA increased bacterial virulence in a murine sepsis model. Our findings help in understanding the adaptive dynamics and strategies of E. coli during short-term natural evolution.

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica , whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade- and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica , including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica .

Halocin S8 is a hydrophobic microhalocin of 36 amino acids (3,580 Da) and is the first microhalocin to be described. This peptide antibiotic is unique since it is processed from inside a much larger, 33,962-Da pro-protein. Halocin S8 is quite robust, as it can be desalted, boiled, subjected to organic solvents, and stored at 4°C for extended periods without losing activity. The complete amino acid sequence of halocin S8 was obtained first by Edman degradation of the purified protein and verified from the halS8 gene: H 2 N-S-D-C-N-I-N-S-N-T-A-A-D-V-I-L-C-F-N-Q-V-G-S-C-A-L-C-S-P-T-L-V-G-G-P-V-P-COOH. The halS8 gene is encoded on an ∼200-kbp megaplasmid and contains a 933-bp open reading frame, of which 108 bp are occupied by halocin S8. Both the halS8 promoter and the “leaderless” halS8 transcript are typically haloarchaeal. Northern blot analysis revealed three halS8 transcripts: two abundant and one minor. Inspection of the 3′ end of the gene showed only a single, weak termination site (5′-TTTAT-3′), suggesting that some processing of the larger transcripts may be involved. Expression of the halS8 gene is growth stage dependent: basal halS8 transcript levels are present in low concentrations during exponential growth but increase ninefold during the transition to stationary phase. Initially, halocin activity parallels halS8 transcript levels very closely. However, when halocin activity plateaus, transcripts remain abundant, suggesting inhibition of translation at this point. Once the culture enters stationary phase, transcripts rapidly return to basal levels.

The European methicillin-resistant Staphylococcus aureus (MRSA) clone ST80-IV has historically dominated community-associated infections in major parts of Europe and is a lineage strongly linked to skin and soft tissue infections. Here, we report the genome sequence of an ST80-IV representative, 11819-97, isolated from a skin infection in Denmark in 1997.

Bacillus anthracis appears to be the most molecularly homogeneous bacterial species known. Extensive surveys of worldwide isolates have revealed vanishingly small amounts of genomic variation. The biological importance of the resting-stage spore may lead to very low evolutionary rates and, perhaps, to the lack of potentially adaptive genetic variation. In contrast to the overall homogeneity, some gene coding regions contain hypervariability that is translated into protein variation. During marker analysis of diverse strains, we have discovered a novel ca. 750-nucleotide open reading frame (ORF) that contains in-frame, variable-number tandem-repeat sequences. Four distinct variable regions exist within vrrB , giving rise to 11 distinct alleles in eight different length categories among B. anthracis strains. This ORF putatively codes for a 241- to 265-amino-acid protein, rich in glutamine (13.2%), glycine (23.4%), and histidine (23.0%). The variable-region amino acids of the vrrB ORF are strongly hydrophilic. Coupled with putative transmembrane domains flanking the variable regions, this suggests a membrane-anchored cytosolic or extracellular location for the putative protein. Sequence analysis of the complete ORFs from three Bacillus cereus strains shows maintenance of the ORF across species boundaries, including strong conservation of the amino acid sequence and the capacity to vary among strains. The presence of 11 different alleles of the vrrB locus is in stark contrast to the near homogeneity of B. anthracis . Evolution of hypervariable genes can negate the lack of genetic variability in species such as B. anthracis and provide select rapid evolution in other more variable species.

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers ( vrrC 1 , vrrC 2 , vrrB 1 , vrrB 2 , and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously ( vrrA ). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (pag) was sequenced from 26 of the most diverse B. anthracis strains to identify potential variation in the toxin and to further our understanding of B. anthracis evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of pag from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the pag corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in B. anthracis has occurred with little or no horizontal transfer between the different strains. Bacillus anthracis is the causative organism of the potentially fatal disease anthrax. Virulent forms of B. anthracis carry two large plasmids, pX01 (ca. 174 kb) and pX02 (ca. 95 kb). Virulence factors include toxin and capsule production, encoded on pX01 and pX02, respectively. The anthrax toxin is composed of three proteinaceous subunits: (i) lethal factor (LF), the toxin component thought to kill host cells by disrupting the mitogen-activated protein kinase pathway (2); (ii) edema factor (EF), an adenylyl cyclase that causes skin edema in the infected host (6); and (iii) protective antigen (PA), which binds to eucaryotic cell surface proteins, forms homoheptamers, and then binds to and internalizes EF and LF.

The first gene to encode a haloarchaeal bacteriocin (halocin H4) has been cloned and sequenced from Haloferax mediterranei R4. Both the signal sequence in the halocin H4 preprotein and the monocistronic halH4 gene have some unusual features. The physiology of halH4 expression reveals that although halH4 transcripts are present at low basal levels during exponential growth, halocin H4 activity first appears as the culture enters stationary phase. As halocin activity levels increase, so do transcript levels, but then activity levels decrease precipitously while transcript levels remain elevated.