Complete genome sequence of Helicobacter pylori B128 7.13 and a single-step method for the generation of unmarked mutations.

BACKGROUND: Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction-modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori, there are relatively few genetic tools for the mutagenesis of this bacterium. MATERIALS AND METHODS: Here, we use PacBio and Illumina sequencing to reveal the complete genome sequence of H pylori B128 7.13. Furthermore, we describe a system to generate markerless and scarless mutations on the H pylori chromosome using the counter-selection marker, galactokinase from Escherichia coli. RESULTS: We show that this mutagenesis strategy can be used to generate in-frame insertions, gene deletions, and multiple independent mutations in B128 7.13. Using the closed genome as a reference, we also report the absence of second site chromosomal mutations and/or rearrangements in our mutagenized strains. We compare the genome sequence of H pylori B128 7.13 with a closely related strain, H pylori B8, and reveal one notable region of difference, which is a 1430 bp insertion encoding a H pylori-specific DUF874 family protein of unknown function. CONCLUSIONS: This article reports the closed genome of the important H pylori B128 7.13 strain and a mutagenesis method that can be adopted by researchers as an alternative strategy to generate isogenic mutants of H pylori in order to further our understanding of this bacterium.

CetZ tubulin-like proteins control archaeal cell shape.

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.

Strabismus surgery in the over 60s--an update.

BACKGROUND: The proportion of individuals above retirement age is expanding and consequently there is increased demand for ophthalmic surgery, including strabismus. In 2001 we published a pilot study reviewing all strabismus operations performed on patients aged 60 and over between 1992 and 1999. Almost a decade later, we repeated the analysis, aiming to identify any longitudinal trends. METHODS: Using the hospital information system, we conducted a retrospective review of all elective strabismus surgery in patients aged 60 years and over, performed by one surgeon (JPL) between January 2000 and September 2008. All operations were under general anesthesia. RESULTS: We identified 237 strabismus operations (9.1% of total operations) that were performed in patients aged 60 or over. This is significantly higher compared with the proportion of elderly patients in the previous study (111 patients, 7% of total operations, P=0.015). Gender distribution was similar to our previous study (105 females, 51% vs 59 females, 53.1%, P=0.814). Ages ranged from 60 to 96 years (mean 69.5 ± 6.8). A decade ago, the more common diagnoses were fourth (22%) and sixth (10%) cranial nerve palsies, consecutive (14%) and secondary (9%) strabismus, whereas recently thyroid eye disease (18%), sixth nerve palsy (13%) and iatrogenic (11%) causes have overtaken the other categories (P=0.011). Adjustable sutures were undertaken in 144 patients (61%). CONCLUSION: Strabismus in elderly patients continues to be related to neurogenic etiologies but thyroid eye disease and iatrogenic causes are on the increase. This is the first 10-year longitudinal analysis to be presented, with the caveat that our data are obtained from a single practice.