Antigenic drift of non-encapsulated Haemophilus influenzae major outer membrane protein P2 in patients with chronic bronchitis is caused by point mutations

The sequence of the gene encoding major outer membrane protein (MOMP) P2 of antigenic variants of non-encapsulated Haemophilus influenzae isolated from persistently infected chronic bronchitis patients was analysed. Antigenic drift was shown to result from single base changes in the P2 gene, all generating amino acid changes in the surface-exposed loops of MOMP P2, predominantly in loop 6. Similar single base changes were observed in H. influenzae persistently present in a subcutaneous cage implanted in rabbits, as well as in a spontaneous H. influenzae mutant that had survived MOMP P2 specific monoclonal-antibody-dependent bactericidal killing in vitro. We hypothesize that accumulation of point mutations under the selection pressure of immunity is a mechanism of antigenic drift of a surface-exposed protein during persistent H. influenzae infection     

Biochemically distinct vesicles from the endoplasmic reticulum fuse to form peroxisomes

As a rule, organelles in eukaryotic cells can derive only from pre-existing organelles. Peroxisomes are unique because they acquire their lipids and membrane proteins from the endoplasmic reticulum (ER), whereas they import their matrix proteins directly from the cytosol. We have discovered that peroxisomes are formed via heterotypic fusion of at least two biochemically distinct preperoxisomal vesicle pools that arise from the ER. These vesicles each carry half a peroxisomal translocon complex. Their fusion initiates assembly of the full peroxisomal translocon and subsequent uptake of enzymes from the cytosol. Our findings demonstrate a remarkable mechanism to maintain biochemical identity of organelles by transporting crucial components via different routes to their final destination.     

Mechanism and control of Genipa americana seed germination

Genipa americana (Rubiaceae) is important for restoration of riparian forest in the Brazilian Cerrado. The objective was to characterize the mechanism and control of germination of G. americana to support uniform seedling production. Morphology and morphometrics of seeds, embryo and endosperm were assessed by light and scanning electron microscopy during germination. Imbibition and germination curves were generated and over the same time interval endosperm digestion and resistance were measured by puncture force analysis and activity assay of endo-β-mannanase (EBM) in water and in abscisic acid (ABA). The gene encoding for EBM was partially cloned and its expression monitored by quantitative real-time-polymerase chain reaction. Embryos displayed growth prior to radicle protrusion. A two-phase increase in EBM activity coincided with the two stages of weakening of the micropylar endosperm. The second stage also coincided with growth of the embryo prior to radicle protrusion. Enzyme activity was initiated in the micropylar endosperm but spread to the lateral endosperm. ABA completely inhibited germination by inhibiting embryo growth, the second stage of weakening and expression of the EBM gene, but EBM activity was not significantly inhibited. This suggests that a specific isoform of the enzyme is involved in endosperm weakening. EBM may cause a general 'softening' of micropylar endosperm cell walls, allowing the embryo to puncture the endosperm as the driving force of the decrease in puncture force.     

Lead, zinc and cadmium accumulation from two metalliferous soils with contrasting calcium contents in heavy metal-hyperaccumulating and non-hyperaccumulating metallophytes: a comparative study

Aims and background

We previously compared metallicolous (M) and non-metallicolous (NM) populations of Noccaea (=Thlaspi) caerulescens, Silene vulgaris, and Matthiola flavida for their abilities to tolerate and (hyper)-accumulate lead (Pb) in hydroponics. In the present study we aimed 1) to check the hyperaccumulation and tolerance abilities of these populations in controlled experiments using metalliferous soils, 2) to test the M. flavida M population for Zn and Cd hypertolerance in hydroponics.

Methods

Plants were grown in hydroponics and fertilized metalliferous substrates, collected from a Zn/Pb smelter sinter deposit near Plombières, Belgium (low pH, low Ca), and a tailing of the Irankouh Zn/Pb mine, Iran (high pH, high Ca). Metal tolerance was assessed from root growth inhibition in hydroponics, or mortality, stunting or chlorosis in the experiments with soil.

Results

Metallicolous M. flavida did not show hypertolerance or hyperaccumulation of Cd or Zn in hydroponics. Only one of the N. caerulescens M populations and the native S. vulgaris M population were able to grow in Plombières soil, whereas the others stopped growing or died within 40?days. All the populations survived and maintained growth for 40?days in Irankouh soil. When grown in Irankouh soil, the M population of M. flavida hyperaccumulated Pb. N. caerulescens hyperaccumulated Zn from Plombières soil, but not from Irankouh soil.

Conclusions

The M. flavida M population is non-Pb-hypertolerant. It hyperaccumulates Pb from Irankouh soil, but not from Pb-amended nutrient solution. N. caerulescens does not hyperaccumulate Zn from the calcareous Irankouh soil.     

Detection and identification of species-specific bacteria associated with synanthropic mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2?×?10(2) to 1.4?×?10(3) per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of "Candidatus Cardinium hertigii" and as a separate novel cluster.     

Bacterial cell surface damage due to centrifugal compaction

Centrifugal damage has been known to alter bacterial cell surface properties and interior structures, including DNA. Very few studies exist on bacterial damage caused by centrifugation because of the difficulty in relating centrifugation speed and container geometry to the damage caused. Here, we provide a simple, versatile method of analysis for describing the compaction of bacteria during centrifugation based on a proposed centrifugation coefficient, C. Values of C can be related to different bacterial cell surface properties. Changing the geometry of the centrifugation container or centrifugation speeds changed the value of C significantly. Initial deposition rates of Staphylococcus aureus ATCC 12600 to a glass surface decayed exponentially from 4,217 to 1,478 cm⁻² s⁻¹ with increasing C, while the proportion of staphylococci with a zeta potential of around −15 mV decreased from 97 to 58%. These surface-sensitive parameters were used independently to derive a critical centrifugation coefficient (0.040), above which centrifugation was considered to impact the outcome of surface-sensitive experiments due to cell surface damage. The critical centrifugation coefficient could successfully predict staphylococcal cell surface damage, i.e., a significant change in initial deposition rate or zeta potential distribution, in 84% of all cases included here, whereas the centrifugation speed could predict damage in only 58% of all cases. Moreover, controlling the centrifugation coefficient within narrow limits over a series of experiments yielded 43% smaller standard deviations in initial staphylococcal deposition rates than with centrifugation at fixed speeds for replicate experiments.     

Adhesive bond stiffness of Staphylococcus aureus with and without proteins that bind to an adsorbed fibronectin film

Staphylococcus aureus is known to cause biomaterial-associated infections of implants and devices once it has breached the skin and mucosal barriers. Adhesion is the initial step in the development of a biomaterial-associated infection, and strategies to prevent staphylococcal adhesion and thus biomaterial-associated infections require understanding of the adhesive bond. The aim of this study was to compare the adhesive bond stiffnesses of two S. aureus strains with and without fibronectin-binding proteins (FnBPs) adhering to a fibronectin-coated quartz crystal microbalance (QCM) sensor surface on the basis of a coupled- resonance model. Both fibronectin adsorption and staphylococcal adhesion were accompanied by negative frequency shifts, regardless of the absence or presence of FnBPs on the staphylococcal cell surfaces. This is the opposite of the positive frequency shifts often observed for other bacterial strains adhering to bare sensor surfaces. Most likely, adhering staphylococci sink into and deform the adsorbed protein layer, creating stiff binding with the sensor surface due to an increased bacterium-substratum contact area. S. aureus 8325-4 possesses FnBPs and yields less negative frequency shifts (Δf) that are further away from the zero-crossing frequency than S. aureus DU5883. This suggests that FnBPs on S. aureus 8325-4 create a stiffer bond to the fibronectin coating than has been observed for S. aureus DU5883. Due to a limited window of observation, as defined by the available resonance frequencies in QCM, we could not determine exact stiffness values.