Nonselective Incorporation into Sporangium of Either “Older” or “Younger” Chromosome of the Vegetative Cell During Sporulation in Bacillus cereus

Employing Bacillus cereus strain 2, we examined the fate of two chromosomes contained in vegetative cells in the course of sporulation. Cytological observations and quantitative estimation of deoxyribonucleic acid (DNA) confirmed the earlier observations that, during the course of sporulation, one of two chromosomes of the vegetative cell was incorporated into the sporangium and the other disappeared into the medium as the result of cell lysis. Log-phase cells, labeled completely with thymine-2-(14)C in the presence of deoxyadenosine, were cultured in the "cold" glucose-glutamate-glycine-salts medium, and culture samples, taken at intervals at successive generations, were subjected to sporulation in glutamate-salts medium. The percentage of radioactivity in the spores separated from each culture remained almost unchanged at nearly 50% and was independent of the number of generations of the preceding culture in the "cold" medium. This suggests that the selective incorporation into the sporangium of either the "older" or "younger" chromosome of a vegetative cell does not occur in the course of spore formation. Some examples of the selective and nonselective behavior of DNA molecules in cellular events in microorganisms are cited.     

Ribonuclease II Activity in a “Presumptive” Ribonuclease II-Deficient Mutant

A mutant strain of Escherichia coli previously thought to possess low levels of ribonuclease II activity has normal levels of ribonuclease II after partial purification of this enzyme from crude extracts.     

Sporulation of the “Thermophilic Anaerobes.”

A reasonable degree of synchrony in the sporulation of Clostridium thermosaccharolyticum 3814 was obtained by using three 10% transfers of 8-hr cultures in a medium containing 0.5% L-arabinose, 0.5% peptone, 0.5% yeast extract, and Gc minerals. Sporulation was stimulated by L-arabinose and L-xylose, but was repressed by glucose, mannose, fructose, and D-pentoses. Sporulating cells were long and thin, whereas repressed cells were shorter and thicker. The optimal pH for sporulation was in the range of pH 5.0 to 5.5. As sporulation continued, the accumulated acetate decreased. Label studies indicated that a significant amount of acetate-2-C¹⁴ was incorporated into the spore lipid. The calcium, phosphorus, and dipicolinic acid (DPA) concentrations on a dry weight basis were 2.55, 2.60, and 7.25%, respectively. The molar ratio of Ca-DPA was 1.47.     

Evidence for “Deleted” or “Silent” gene homozygous at the locus coding for the constant region of the γ3 chain

Three uncommon stable Gm haplotypes, Gm3;23;--, Gm1,2,17;..;-- and Gm1,17;..;-- have been transmitted through 3 generations of two related Lebanese and Syrian families. No pathological consequence was noted in seven individuals, aged 14--65, whose sera were deficient for all the allotypes carried by the IgG3 chains. Among the different genetic events which could have produced these haplotypes (alteration of a regulatory gene, point mutation, gene hybridization, gene deletion), it appears that a structural deletion is the most probable explanation. The observed data can be explained by either a partial or a total deletion of the constant portion of the IgG3 heavy chain.     

“Candidatus Endobugula glebosa,” a Specific Bacterial Symbiont of the Marine Bryozoan Bugula simplex

The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, “Candidatus Endobugula sertula.” “Candidatus E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “Candidatus E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status “Candidatus Endobugula glebosa” is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.     

Absence of Direct Delivery for Single Transmembrane Apical Proteins or Their “Secretory” Forms in Polarized Hepatic Cells

The absence of a direct route to the apical plasma membrane (PM) for single transmembrane domain (TMD) proteins in polarized hepatic cells has been inferred but never directly demonstrated. The genes encoding three pairs of apical PM proteins, whose extracellular domains are targeted exclusively to the apical milieu in Madin-Darby canine kidney cells, were packaged into recombinant adenovirus and delivered to WIF-B cells in vitro and liver hepatocytes in vivo. By immunofluorescence and pulse-chase metabolic labeling, we found that the soluble constructs were overwhelmingly secreted into the basolateral milieu, which in vivo is the blood and in vitro is the culture medium. The full-length proteins were first delivered to the basolateral surface but then concentrated in the apical PM. Our results imply that hepatic cells lack trans-Golgi network (TGN)-based machinery for directly sorting single transmembrane domain apical proteins and raise interesting questions about current models of PM protein sorting in polarized and nonpolarized cells.     

Escherichia coli MazF Leads to the Simultaneous Selective Synthesis of Both “Death Proteins” and “Survival Proteins”

The Escherichia coli mazEF module is one of the most thoroughly studied toxin–antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful.     

Genetic Diversity of Porphyromonas gingivalis Isolates Recovered from Single “Refractory” Periodontitis Sites

Multilocus sequence typing and fimA genotyping were performed on Porphyromonas gingivalis isolates from 15 subjects with “refractory” periodontitis. Several sequence types were detected for most individual pockets. The variation indicated recombination at the recA and pepO genes. The prevalence of fimA genotypes II and IV confirmed their association with periodontitis.