SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14

Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state. Here we report a new class of P. aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation. The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P. aeruginosa PA14 and encodes a protein of unknown function. Complementation analysis and phage-mediated transduction demonstrated that the transposon insertion in PA5346 was the cause of the biofilm-defective phenotype. Examination of flow cell-grown biofilms showed that the sadB199 mutant could initiate surface attachment but failed to form microcolonies despite being proficient in both twitching and swimming motility. Closer examination of early attachment revealed an increased number of the sadB199 mutant cells arrested at reversible attachment, functionally defined as adherence via the cell pole. A positive correlation among biofilm formation, irreversible attachment, and SadB level was demonstrated, and furthermore, RpoN and FleR appear to negatively affect SadB levels. Fractionation studies showed that the SadB protein is localized to the cytoplasm, and with the use of GPS-linker scanning mutagenesis, the C-terminal portion of SadB was shown to be dispensable for function, whereas the two putative domains of unknown function and the linker region spanning these domains were required for function. We discuss the results presented here in the context of microbial development as it applies to biofilm formation.     

Xanthine dehydrogenase and aldehyde oxidase impact plant hormone homeostasis and affect fruit size in ‘Hass’ avocado

The contribution of xanthine dehydrogenase (XDH, EC 1.1.1.204) to fruit size was investigated using the normal and small-fruit variants of Persea americana Mill. cv. Hass. Inhibition of XDH by treatment of normal fruit, in the linear phase of growth (phase II), with allopurinol (Allo) arrested fruit growth. Adenine (Ade), a less effective inhibitor of this enzyme, also arrested fruit growth when applied in phase II and slowed fruit growth when applied in phase III. A time-course study on the activity of XDH in mesocarp tissue from normal and small fruit showed that maximum activity occurred late in phase II and that the peak in activity was absent in mesocarp of the small fruit. Feeding Ade to growing fruit in phase III caused a transient decline in fruit growth (measured as change in fruit length). Thereafter, growth resumed although fruit size was irreversibly affected. Treatment of fruit with Ade and Ade-containing cytokinins altered activity of another molybdenum enzyme, aldehyde oxidase (EC 1.2.3.1). Cytokinin oxidase was induced by cytokinin and auxin. Purine catabolism via hypoxanthine/xanthine was operative in normal fruit and in mesocarp from the small-fruit variant and as expected, Allo treatment caused accumulation of xanthine and adenine. In the absence of an increase in XDH during growth of the small-fruit phenotype, low levels of Ade were interpreted as resulting from respiration-enhanced adenylate depletion. Stress and/or pathogen induction of the alternative oxidase pathway is proposed as a possible cause.     

Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

ABSTRACT: BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.     

Prevalence, causes and socio-economic determinants of vision loss in Cape Town, South Africa

Purpose

To estimate the prevalence and causes of blindness and visual impairment in Cape Town, South Africa and to explore socio-economic and demographic predictors of vision loss in this setting.

Methods

A cross sectional population-based survey was conducted in Cape Town. Eighty-two clusters were selected using probability proportionate to size sampling. Within each cluster 35 or 40 people aged 50 years and above were selected using compact segment sampling. Visual acuity of participants was assessed and eyes with a visual acuity less than 6/18 were examined by an ophthalmologist to determine the cause of vision loss. Demographic data (age, gender and education) were collected and a socio-economic status (SES) index was created using principal components analysis.

Results

Out of 3100 eligible people, 2750 (89%) were examined. The sample prevalence of bilateral blindness (presenting visual acuity <3/60) was 1.4% (95% CI 0.9–1.8). Posterior segment diseases accounted for 65% of blindness and cataract was responsible for 27%. The prevalence of vision loss was highest among people over 80 years (odds ratio (OR) 6.9 95% CI 4.6–10.6), those in the poorest SES group (OR 3.9 95% CI 2.2–6.7) and people with no formal education (OR 5.4 95% CI 1.7–16.6). Cataract surgical coverage was 68% in the poorest SES tertile (68%) compared to 93% in the medium and 100% in the highest tertile.

Conclusions

The prevalence of blindness among people ≥50 years in Cape Town was lower than expected and the contribution of posterior segment diseases higher than previously reported in South Africa and Sub Saharan Africa. There were clear socio-economic disparities in prevalence of vision loss and cataract surgical coverage in this setting which need to be addressed in blindness prevention programs.     

Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation

Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100–200 nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment.     

A novel peptide tag for detection and purification of recombinant expressed proteins

Peptide tags have proven useful for the detection and purification of recombinant proteins. However cross reactions of antibodies raised to the tag are frequently observed due to the presence of host proteins containing all or parts of the tag. In this report we have identified a unique viral peptide sequence, R-tag, that by blast searches is absent from the commonly expression hosts Arabidopsis thaliana, Escherichia coli, Pichia pastoris and mouse myeloma cell NSO. We have prepared monoclonal antibodies to this peptide and confirmed the absence of this peptide sequence from the above genomes by Western blotting. We have also modified protein expression vectors to incorporate this sequence as a fusion tag in expressed proteins and shown its use to successfully purify recombinant proteins by immunoaffinity procedures.