Genetic analysis of double-strand break repair in Escherichia coli

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.     

Production of docosahexaenoic and docosapentaenoic acids bySchizochytrium sp. isolated from Yap Islands

A marine microbe (strain SR21) from the coral reef area of the Yap Islands was isolated by a screening test for polyunsaturated fatty acids and was found to accumulate lipid that contained 22:5n-6 docosapentaenoic acid (DPA) as well as 22:6n-3 docosahexaenoic acid (DHA). Strain SR21 was identified as genusSchizochytrium in Labyrinthulomycota, owing to its ultrastructural character and life cycle, which is composed of vegetative cell, zoosporangium, and zoospore stages. After cultural optimization, both in flask and fermenter, the highest DHA and DPA productivities of 2.0 and 0.44 g/L per day, respectively, were obtained in a medium of 60 g/L glucose and corn steep liquor/ammonium sulfate in a half salt concentration of seawater in fermenter culture at 28°C and pH 4. This productivity was almost twice that obtained with flask culture, indicating its high resistance to mechanical stirring. The lipid extracted from the cell was about 50% of the dry cell weight and was composed of 93% triacylglycerol (TG). DHA content of the lipid was 34% of total fatty acids. The TG profile was simple, and the content of the most dominant TG, 1,3-dipalmitoyl-2-DHA-TG, was 27%. TG that contained DHA and n-6-DPA amounted to 57 and 17%, respectively, of total TG molecules. Strain SR21 was revealed to be an excellent source of microbial DHA and n-6 DPA.     

Pathologic and biochemical studies of juvenile parkinsonism linked to chromosome 6q

We report the results of pathologic and biochemical studies in a patient with 6q-linked autosomal recessive juvenile parkinsonism (AR-JP). Neuronal loss and gliosis were restricted to the substantia nigra and the locus ceruleus. No Lewy bodies were found, but neurofibrillary tangles and argyrophilic astrocytes were seen in the cerebral cortex and brainstem nuclei. The later findings, which have not been reported previously in AR-JP, suggest the pathologic heterogeneity of 6q-linked AR-JP.     

Steam reformation of hydrogen sulfide

A modified version of the Sulfur–Iodine cycle, here called the Sulfur–Sulfur Cycle, offers an all-fluid route to thermochemical hydrogen and avoids implications of the corrosive HI–H₂O azeotropic mixture:

equation(1)

4I_2(l) + 4SO_2(l) + 8H₂O_(l) ↔ 4H₂SO_4(l) + 8HI_(l) (120 °C)

equation(2)

8HI_(l) + H₂SO_4(l) ↔ H₂S_(g) + 4H₂O_(l) + 4I_2(l) (120 °C)

equation(3)

3H₂SO_4(g) ↔ 3H₂O_(g)+3SO_2(g) + 1½O_2(g) (850 °C)

equation(4)

H₂S_(g) + 2H₂O_(g) ↔ SO_2(g) + 3H_2(g) (900–1500 °C)

     

Profile of polyunsaturated fatty acids produced by Thraustochytrium sp. KK17-3

More than 300 strains of microorganisms producing polyunsaturated fatty acids (PUFA) were newly isolated from coastal seawater in the Seto Inland Sea and around Iriomote Island, Japan, by the baiting method. The profiles of PUFA from docosahexaenoic acid (DHA)-producing strains could be classified into four types. A strain, named KK17-3, was chosen for further study owing to its high DHA content (52.1% of total fatty acid) and wide range of PUFA (76.1%) including arachidonic, eicosapentaenoic, and docosapentaenoic acids as well as DHA. Glucose and tryptone were the optimal carbon and nitrogen sources, respectively, in a medium with salinity at 75% that of seawater. The PUFA contents in polar lipids (22.1% of total lipid), in which the DHA content was 39.3%, were higher than those in neutral lipids and glycolipids. Molecular phylogenetic analysis of 18S rRNA gene sequences showed KK17-3 to be a thraustochytrid. It also was observed to possess a life cycle composed of vegetative cells without successive bipartition, zoosporangium, and zoospore stage. Classification by the chemotaxonomic criterion based on PUFA compositions also supported this assignment.     

Low-level release of Shiga-like toxin (verocytotoxin) and endotoxin from enterohemorrhagic Escherichia coli treated with imipenem

Shiga-like toxin (SLT) and endotoxin may participate in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC) infection. Levels of release of SLT and endotoxin from EHEC treated in vitro with antibiotics were estimated. There were differential levels of release of SLT and endotoxin from EHEC treated with different antibiotics. Treatment of EHEC strains, namely, E. coli O157, O111, and O26, with imipenem induced much lower levels of release of SLT and endotoxin than treatment with ceftazidime.     

Method for purification of krypton from environmental samples for analysis of radiokrypton isotopes

Radiokrypton isotopes ((81)Kr and (85)Kr) are ideal tracers and chronometers of various environmental processes. Atom trap trace analysis (ATTA) is capable of determining the ultralow isotopic abundances of radiokryptons (<10(-12)) provided that 50 microL of pure Kr is available. The analysis by using ATTA of (81)Kr in naturally occurring gases of interest, e.g., dissolved gases in hydrological reservoirs, requires separation of parts-per-million (ppm) level Kr from chemically airlike bulk gas. A newly developed Kr purification system is based on conventional cryogenic distillation and gas chromatography to which continuous monitoring of gas effluent composition using a quadrupole mass spectrometer brings significant advantages. Simple cryogenic distillation is controlled based on the evolution of N2/Ar ratio that is relatively constant in naturally occurring, inorganic gas. Gas chromatographic separation of parts-per-million by volume (ppmv) level Kr from up to a few liters of bulk gas can be achieved by concentrating the Kr under the chromatographic tails of major components. The system described here is capable of extracting Kr of >98% purity from 5-125 L STP (standard temperature and pressure) of bulk gas with >90% yield within several hours. This system is generally useful for separation of microliter amounts of unreactive trace volatile compounds from large-volume gas samples.     

Differential release of smooth-type lipopolysaccharide from Pseudomonas aeruginosa treated with carbapenem antibiotics and its relation to production of tumor necrosis factor alpha and nitric oxide

Endotoxin release from Pseudomonas aeruginosa treated with cell wall-active carbapenem antibiotics and its effect on the production of tumor necrosis factor alpha and nitric oxide were examined. Treatment of bacteria with imipenem induced much lower levels of endotoxin release than treatment with meropenem. The endotoxin released was demonstrated to be of the smooth type and O-specific polysaccharide-rich. The exposure of the filtrates of P. aeruginosa treated with imipenem to physiologically relevant cells caused low-level production of tumor necrosis factor alpha and nitric oxide, while similar treatment with meropenem induced high levels of production.     

Production of high yields of docosahexaenoic acid by Schizochytrium sp. strain SR21

The culture conditions for high-yield production of docosahexaenoic acid (DHA) by Schizochytrium sp. strain SR21 were investigated in a fermenter. With increasing carbon (glucose) and nitrogen (corn steep liquor and ammonium sulfate) sources (up to 12% glucose) in the medium, DHA productivity increased without a decrease in growth rate, i.e., 2.0, 2.7, and 3.3 g DHA/L/d with 6, 10, and 12% glucose, respectively. Eventually, 48.1 g dry cells/L and 13.3 g DHA/L were produced in 4 d with 12% glucose. DHA productivity was decreased with 15% glucose, i.e., 3.1 g/L/d. With 12% glucose, the lipid content was 77.5% of dry cells, and DHA content was 35.6% of total fatty acids. The lipid was composed of about 95% neutral lipid and 5% polar lipid. In polar lipids, the contents of phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylinositol were 74, 11, and 5%, respectively. The PC profile was simple, 70% of PC molecules were 1-palmitoyl-2-DHA-PC and 1.2-di-DHA-PC. These results indicate that Schizochytrium sp. strain 21 is an excellent source for microbial DHA production, including not only the acid form of DHA but also 2-DHA-PC.