Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil

Bacillus is commonly isolated from soils, with organisms of Bacillus cereus sensu lato being prevalent. Knowledge of the ecology of B. cereus and other Bacillus species in soil is far from complete. While the older literature favors a model of growth on soil-associated organic matter, the current paradigm is that B. cereus sensu lato germinates and grows in association with animals or plants, resulting in either symbiotic or pathogenic interactions. An in terra approach to study soil-associated bacteria is described, using filter-sterilized soil-extracted soluble organic matter (SESOM) and artificial soil microcosms (ASM) saturated with SESOM. B. cereus ATCC 14579 displayed a life cycle, with the ability to germinate, grow, and subsequently sporulate in both the liquid SESOM extract and in ASM inserted into wells in agar medium. Cells grew in liquid SESOM without separating, forming multicellular structures that coalesced to form clumps and encasing the ensuing spores in an extracellular matrix. Bacillus was able to translocate from the point of inoculation through soil microcosms as shown by the emergence of outgrowths on the surrounding agar surface. Microscopic inspection revealed bundles of parallel chains inside the soil. The motility inhibitor L-ethionine failed to suppress outgrowth, ruling out translocation by a flagellar-mediated mechanism such as swimming or swarming. Bacillus subtilis subsp. subtilis Marburg and four Bacillus isolates taken at random from soils also displayed a life cycle in SESOM and ASM and were all able to translocate through ASM, even in presence of L-ethionine. These data indicate that B. cereus is a saprophytic bacterium that is able to grow in soil and furthermore that it is adapted to translocate by employing a multicellular mode of growth.     

Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte

O-Linked N-acetylglucosaminylation (O-GlcNAcylation) (or O-linked N-acetylglucosamine (O-GlcNAc)) is an abundant and reversible glycosylation type found within the cytosolic and the nuclear compartments. We have described previously the sudden O-GlcNAcylation increase occurring during the Xenopus laevis oocyte G(2)/M transition, and we have demonstrated that the inhibition of O-GlcNAc-transferase (OGT) blocked this process, showing that the O-GlcNAcylation dynamism interferes with the cell cycle progression. In this work, we identified proteins that are O-GlcNAc-modified during the G(2)/M transition. Because of a low expression of O-GlcNAcylation in Xenopus oocyte, classical enrichment of O-GlcNAc-bearing proteins using O-GlcNAc-directed antibodies or wheat germ agglutinin lectin affinity were hard to apply, albeit these techniques allowed the identification of actin and erk2. Therefore, another strategy based on an in vitro enzymatic labeling of O-GlcNAc residues with azido-GalNAc followed by a chemical addition of a biotin alkyne probe and by enrichment of the tagged proteins on avidin beads was used. Bound proteins were analyzed by nano-LC-nano-ESI-MS/MS allowing for the identification of an average of 20 X. laevis oocyte O-GlcNAcylated proteins. In addition to actin and beta-tubulin, we identified metabolic/functional proteins such as PP2A, proliferating cell nuclear antigen, transitional endoplasmic reticulum ATPase, aldolase, lactate dehydrogenase, and ribosomal proteins. This labeling allowed for the mapping of a major O-GlcNAcylation site within the 318-324 region of beta-actin. Furthermore immunofluorescence microscopy enabled the direct visualization of O-GlcNAcylation and OGT on the meiotic spindle as well as the observation that chromosomally bound proteins were enriched in O-GlcNAc and OGT. The biological relevance of this post-translational modification both on microtubules and on chromosomes remains to be determined. However, the mapping of the O-GlcNAcylation sites will help to underline the function of this post-translational modification on each identified protein and will provide a better understanding of O-GlcNAcylation in the control of the cell cycle.     

Biofilm proteome: homogeneity or versatility?

The problems associated with biofilm infections in humans result from the distinct characteristics of biofilms, in particular their high level of resistance to antibiotics. One of the hypotheses that have been advanced to explain this resistance to antimicrobials is the phenotypic differentiation of biofilm cells. Although many studies on biofilms have highlighted physiological alterations following the attachment of bacteria to a surface, no studies have explicitly demonstrated a "biofilm" physiology. To contribute to this topical debate, we used principal component analysis to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis of crude protein extracts from planktonic and sessile Pseudomonas aeruginosa cells. These analyses showed that the proteome of attached P. aeruginosa cells differs from that of their planktonic counterparts. Furthermore, we found that the proteome of sessile P. aeruginosa is strongly dependent on the nature of the biofilm substratum.     

The chromosome 11 region from strain 129 provides protection from sex reversal in XYPOS mice

C57BL/6J (B6) mice containing the Mus domesticus poschiavinus Y chromosome, YPOS, develop ovarian tissue, whereas testicular tissue develops in DBA/2J or 129S1/SvImJ (129) mice containing the YPOS chromosome. To identify genes involved in sex determination, we used a congenic strain approach to determine which chromosomal regions from 129Sl/SvImJ provide protection against sex reversal in XYPOS mice of the C57BL/6J.129-YPOS strain. Genome scans using microsatellite and SNP markers identified a chromosome 11 region of 129 origin in C57BL/6J.129-YPOS mice. To determine if this region influenced testis development in XYPOS mice, two strains of C57BL/6J-YPOS mice were produced and used in genetic experiments. XYPOS adults homozygous for the 129 region had a lower incidence of sex reversal than XYPOS adults homozygous for the B6 region. In addition, many homozygous 129 XYPOS fetuses developed normal-appearing testes, an occurrence never observed in XYPOS mice of the C57BL/6J-YPOS strain. Finally, the amount of testicular tissue observed in ovotestes of heterozygous 129/B6 XYPOS fetuses was greater than the amount observed in ovotestes of homozygous B6 XYPOS fetuses. We conclude that a chromosome 11 locus derived from 129Sl/SvImJ essentially protects against sex reversal in XYPOS mice. A number of genes located in this chromosome 11 region are discussed as potential candidates.     

Interference of the PAF-acether antagonist BN 52021 with passive anaphylaxis in the guinea-pig

PAF-acether may be involved in anaphylaxis and asthma. We tested the new PAF-acether antagonist BN 52021 against the effects of antigen in passively sensitized guinea-pigs. Bronchoconstriction by ovalbumin administered i.v. (1 mg/kg) or by aerosol (1 or 10 mg/ml for a period of 1 min) was significantly reduced by BN 52021 (1-10 mg/kg), which did not inhibit drop of leukocyte counts after the i.v. challenge. In both cases, when the guinea-pigs were pretreated by propranolol, high amounts of BN 52021 became ineffective against shock. The reduction of the anaphylactic bronchoconstriction, induced by the combination of mepyramine, aspirin and FPL 55712 was not improved by BN 52021. Tested on isolated lung strips from passively sensitized guinea-pig, BN 52021, at a concentration which inhibits PAF-induced contraction (0.1 mM), did not inhibit the anaphylactic contraction triggered by the administration of ovalbumin (10 micrograms/ml) nor the accompanying release of histamine and thromboxane. In contrast, BN 52021 (30 microM) significantly reduced the anaphylactic release of histamine and of thromboxane from perfused lungs of passively sensitized guinea-pigs. The results with the isolated lung strips and the propranolol-treated guinea-pigs in vivo suggest a dissociation between the anti-anaphylactic and the anti-PAF-acether properties of BN 52021.     

Familial case with sequence variant in the testis-determining region associated with two sex phenotypes.

The human Y chromosome encodes a testis-determining factor (TDF) which is responsible for initiating male sex determination. Recently a region of the Y chromosome (SRY) was identified as part of the TDF gene. We have identified a three-generation family (N) in which all XY individuals have a single base-pair substitution resulting in a conservative amino acid change in the conserved domain of the SRY open reading frame. Three individuals are XY sex-reversed females, and two are XY males. Several models are proposed to explain association between a sequence variant in SRY and two sex phenotypes.