Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168

Summary A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.     

Disruption of the langerin/CD207 gene abolishes Birbeck granules without a marked loss of Langerhans cell function

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.     

External mouthpart morphology in the Tenuipalpidae (Tetranychoidea): Raoiella a case study

The use of low-temperature scanning electron microscopy (LTSEM) to study external mouthpart morphology in the Tenuipalpidae, in particular the genus Raoiella, has brought some aspects of the mechanics of feeding in this group into question. In addition, an LTSEM study on the specialized feeding behaviour of Raoiella indica Hirst (Tetranychoidea: Tenuipalpidae) revealed host plant use in this species could be affected by stomatal complex morphology.     

An IP3R3- and NPY-Expressing Microvillous Cell Mediates Tissue Homeostasis and Regeneration in the Mouse Olfactory Epithelium

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3^+/−, and IP3R3^−/− mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3^−/− mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3^−/− mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3^−/− mice. The reductions in both NPY release and number of progenitor cells in IP3R3^−/− mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.     

Relationship Between the Location of Autolysin, Cell Wall Synthesis, and the Development of Resistance to Cellular Autolysis in Streptococcus faecalis After Inhibition of Protein Synthesis

Ten minutes after inhibition of protein synthesis with chloramphenicol (CAP) the ability of cells of Streptococcus faecalis (ATCC 9790) to autolyze decreased to less than 20% of the rate for exponential-phase cells. After threonine exhaustion, the time for a 50% drop in the rate of cellular autolysis was about 20 min. These rapid increases in resistance to cellular autolysis could not be accounted for by: (i) the relatively slow and small overall decrease in susceptibility of isolated cell walls to added autolysin, or (ii) a decreased content of either the active or latent (proteinase activatable) form of the autolysin in the wall fraction. Continued wall synthesis resulted in dilution of preexisting autolysin in the isolated wall fraction. The release of labeled "old" relative to "new" wall from CAP-treated cultures showed that wall synthesis shifted away from the areas of wall previously shown to be associated with wall synthesis (extension) in exponential-phase cells. A corresponding dispersal of active autolysin activity was not observed. By using actinomycin D and CAP, a requirement for ribonucleic acid and protein synthesis early in the recovery of cells from amino acid starvation was demonstrated for the recovery in the ability of cells to autolyze. Evidence was obtained which suggests that a protein is involved in the conversion of latent to active autolysin. During recovery from amino acid starvation, increase in wall synthesis and content of active autolysin was delayed (25 to 35 min), whereas an increase in turbidity and latent enzyme content began within 10 min. After treatment with CAP at 22 or 52 min of recovery, a further increase in levels of both active and latent autolysin was severely inhibited; however, the increase in rate of wall synthesis was indistinguishable from that of an untreated control. This suggests that an increase in rate of wall synthesis does not depend on an increase in level of active autolysin.     

Effect of salt on aerobic biodegradation of petroleum hydrocarbons in contaminated groundwater

Hydrocarbon-contaminated soil and groundwater at oil and gas production sites may be additionally impacted by salts due to release of produced waters. However, little is known about the effect of salt on the in-situ biodegradation of hydrocarbons by terrestrial microbes, especially at low temperatures. To study this effect, we prepared a groundwater-soil slurry from two sites in Canada: a former flare pit site contaminated with flare pit residue (Site A), and a natural gas processing facility contaminated with natural gas condensate (Site B). The slurry with its indigenous microbes was amended with radiolabeled hydrocarbons dissolved in free product plus nutrients and/or NaCl, and incubated in aerobic biometer flasks with gyrotory shaking at either 25 or 10°C for up to 5 weeks. Cumulative production of ¹⁴CO₂ was measured and the lag time, rate and extent of mineralization were calculated. For Site A, concentrations of NaCl ≥1% (w/v) delayed the onset of mineralization of both ¹⁴C-hexadecane and ¹⁴C-phenanthrene under nutrient-amended conditions, but once biodegradation began the degradation rates were similar over the range of salt concentrations tested (0–5% NaCl). For Site B, increasing concentrations of NaCl ≥1% (w/v) increased the lag time and decreased the rate and extent of mineralization of aliphatic and aromatic substrates. Of particular interest is the observation that low concentrations of salt (≤1% NaCl) slightly stimulated mineralization in some cases.     

Relationship between the Latent Form and the Active Form of the Autolytic Enzyme of Streptococcus faecalis

A 10-hr starvation of Streptococcus faecalis ATCC 9790 for the amino acids methionine and threonine results in cells which are resistant to autolysis and which contain greatly reduced quantities of both active and latent (proteinase activable) forms of the autolytic enzyme (an N-acetyl-muramide glycanhydrolase). Cell walls were isolated from cells harvested at various times during the recovery from such starvation and were assayed for active and latent forms of the autolysin. Within 10 min of recovery the latent enzyme began to increase. Only after 30 to 60 min did the active enzyme begin to increase; after a similar lag, the cells' proneness to lysis markedly increased. The intracellular localization of both forms of the autolysin was examined, using as an experimental tool the ability of added cell wall to bind autolysin. (14)C-lysine-labeled, inactivated cell walls were added to exponential-phase cells, which were then disrupted, and the mixed wall population was isolated. Measurement of the (14)C release during wall autolysis indicated that the active enzyme in the cells was not available for binding to the added (14)C-labeled walls and was therefore wall-bound in vivo. In contrast, up to 85% of latent autolysin activity was found to have been efficiently bound to the added (14)C walls. The results obtained suggest (i) cellular autolysis is a reflection of the level of active enzyme and not of latent enzyme, and (ii) autolysin is synthesized and mainly located in the cytoplasm as an inactive latent precursor (proenzyme) which is transported to sites on the cell wall associated with wall biosynthesis, where it becomes activated.