Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome II.

Rhodobacter sphaeroides 2.4.1T is a purple nonsulfur facultative phototrophic bacterium which exhibits remarkable metabolic diversity as well as genomic complexity. Under anoxic conditions, in the absence of light and the presence of dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), R. sphaeroides 2.4.1T utilizes DMSO or TMAO as the terminal electron acceptor for anaerobic respiration, which is mediated by the molybdoenzyme DMSO reductase. Sequencing of a 13-kb region of chromosome II revealed the presence of 10 putative open reading frames, of which 5 possess homology to genes encoding the TMAO reductase (the tor system) of Escherichia coli. The dorS and dorR genes encode a sensor-regulator pair of the two-component sensory transduction protein family, homologous to the torS and torR gene products. The dorC gene was shown to encode a 44-kDa DMSO-inducible c-type cytochrome. The dorB gene encodes a membrane protein of unknown function homologous to the torD gene product. The dorA gene encodes DMSO reductase, containing the molybdopterin active site. Mutations were constructed in each of these dor genes, and the resulting mutants were shown to be impaired for DMSO-dependent anaerobic growth in the dark. The mutant strains exhibited negligible levels of DMSO reductase activity compared to the wild-type strain under similar growth conditions. Further, no DorA protein was detected in DorS and DorR mutant strains with anti-DorA antisera, suggesting that the products of these genes are required for the positive regulation of dor expression in response to DMSO. This characterization of the dor gene cluster is the first evidence that genes of chromosome CII encode metabolic functions which are essential under particular growth conditions.     

Sequence Tag Site and Host Range Assays Demonstrate that Radapholus similis and R. citraphilus are not Reproductively Isolated

Males of citrus-parasitic Radopholus citrophilus (FL1) were mated with non-citrus-parasitic R. similis (FL5) females. Progeny inherited a 2.4-kb sequence tag site (DK#1) and the ability to reproduce in citrus from the paternal parent (FLl); both traits were absent in the maternal line (FL5). The hybrid progeny produced offspring in roots of citrus seedlings over an 8-month period and therefore were considered reproductively viable. Genomic DNA hybridization studies indicated that one or more copies of DK#1 were present in R. citrophilus FL1. It is not likely that DK#1 represents a citrus parasitism gene because it was amplified from some burrowing nematode isolates that did not parasitize citrus and because DK#1 contains no open reading frames. Inability to reliably test individual nematodes for their ability to parasitize citrus was a constraint to obtaining F2 data required for definitive genetic characterization of citrus parasitism in burrowing nematodes, and alternate approaches will be required. Although the physical relationship of DK#1 and the citrus parasitism locus remains undefined, results of controlled mating studies using these parameters as genetic markers enabled us to identify hybrid F₁ progeny. Therefore, R. similis and R. citrophilus are not sibling species since gene flow between the two does not appear to be restricted via geographic isolation (sympatric in Florida) or by genetics.     

Endogenous prostaglandin secretion during cloprostenol-induced abortion in mares

Repeated administration of prostaglandin is the treatment of choice for the termination of pregnancy in mares more than 40 days pregnant. Even though it is well documented that PGF-2 or analogue needs to be administered every 12–24 h for successful induction of abortion, little is known about the underlying endocrine changes and the mechanism by which abortion occurs. The aim of this study was to characterize the changes in PGF-2, progesterone and estrogen secretion during prostaglandin-induced abortion. Six mares, 82–102 days pregnant, were treated daily with 250 μg cloprostenol, blood was collected at 1-h intervals until fetal expulsion and pregnancy examination was performed daily. Four mares, 92–97 days pregnant, received no treatment but were subjected to the same hourly blood collections and daily genital examinations described for cloprostenol-treated mares for 3 days. Mean time from first cloprostenol administration until fetal expulsion was 48.6 ± 5.6 h and required 2.8 ± 0.2 cloprostenol administrations. In all mares, progesterone concentrations decreased in a near linear manner after the first cloprostenol administration and were invariably low (1.3 ± 0.2 ng ml⁻¹, mean ± SEM) at the time of fetal expulsion. Mean estrogen secretion remained unchanged until 5 h before fetal expulsion and then decreased rapidly to non-pregnant levels. Endogenous PGF-2 secretion rate increased with each cloprostenol administration and culminated in sustained PGF-2 secretion which persisted until fetal expulsion was completed. From these results we conclude that cloprostenol-induced abortion is associated with endogenous PGF-2 secretion, fetal expulsion coincides with sustained PGF-2 secretion and low progesterone concentrations and plasma estrogen concentrations remain unchanged until hours before fetal expulsion.     

Benthic bacterial biomass supported by streamwater dissolved organic matter

Bacterial biomass in surface sediments of a headwater stream was measured as a function of dissolved organic carbon (DOC) flux and temperature. Bacterial biomass was estimated using epifluorescence microscopic counts (EMC) and ATP determinations during exposure to streamwater containing 1,788g DOC/liter and after transfer to groundwater containing 693g DOC/liter. Numbers of bacteria and ATP concentrations averaged 1.36×10⁹ cells and 1,064 ng per gram dry sediment, respectively, under initial DOC exposure. After transfer to low DOC water, biomass estimates dropped by 53 and 55% from EMC and ATP, respectively. The decline to a new steady state occurred within 4 days from ATP assays and within 11 days from EMC measures. A 4°C difference during these exposures had little effect on generation times. The experiment indicated that 27.59 mg/hour of natural DOC supported a steady state bacterial biomass of approximately 10g C/g dry weight of sediment (from EMC determinations). Steady state bacterial biomass estimates on sediments that were previously muffled to remove organic matter were approximately 20-fold lower. The ratio of GTPATP indicated differences in physiological condition or community composition between natural and muffled sediments.     

Rapid and early determination of sex using trophoblast biopsy specimens and Y chromosome specific DNA probes.

The feasibility of determining sex by analysing deoxyribonucleic acid (DNA) with two probes specific for Y chromosomes was shown using DNA obtained from samples of blood from 30 non-related males and females of different ethnic origin. The DNA was spotted on nitrocellulose filters and hybridised with both a repetitive (P1) and a unique (49f) sequence specific for the human Y chromosome. A strong positive signal with both probes indicated the presence of male DNA. The sex of 12 fetuses was then similarly determined by molecular characterisation of DNA from trophoblast biopsy specimens. Chorionic samples were obtained in seven cases before termination of pregnancy in the first trimester and the aborted embryos subjected to karyotyping and sex chromatin analysis. In the five other cases samples were obtained from placentas obtained during caesarean section. Results of hybridisation were compared with those from cytogenic studies and actual sex at birth. The sex of all 12 fetuses was determined correctly by hybridisation.     

Accumulation of nucleotides by starved Escherichia coli cells as a probe for the involvement of ribonucleases in ribonucleic acid degradation.

The acid-soluble ribonucleic acid degradation products formed by Escherichia coli cells starved for a carbon source have been identified. They comprise oligonucleotides, nucleoside diphosphates, 5'- and 3'-nucleoside monophosphates, nucleosides, and free bases. The majority of these products are excreted phates, nucleosides, and free bases. The majority of these products are excreted into the medium, and only small and constant amounts are kept in the pool. During carbon starvation at elevated temperatures, mutants deficient in ribonuclease I do not form oligonucleotides and 3'-nucleoside monophosphates, and mutants that contain a modified form of polynucleotide phosphorylase do not accumulate nucleoside diphosphates. 5'-Nucleoside monophosphates do accumulate, however, in a mutant containing thermoabile ribonuclease II, under conditions where more than 95% of all enzyme activity had been destroyed. The data presented confirm the participation of ribonuclease I and polynucleotide phosphorylase in the final steps of ribonucleic acid degradation and indicate that an exonuclease forming 5'-nucleoside monophosphates is also involved.     

Schizogony in Haemoproteus columbae Kruse.

To fill in some of the gaps in our knowledge of Schizogony of Haemoproteus columbae Kruse, transmission experiments involving inoculation into pigeons (Columba livia Gmelin) of sporozonites from salivary glands of the hippoboscid fly Pseudolynchia canariensis (Macquart) were carried out. We were unable to detect prepatent schizonts or to observe schizogonic development when infection became chronic. Schizonts were mainly confined to lung tissue. Observations of parapatent schizonts were made in smears and tissue sections. A variety of forms was found. Cytomeres were rarely encountered. Two types of morphologically distinct merozoites were seen. One type was twice as large as large as the other and was thought to repeat the process of schizogony several times before invading erythrocytes. Schizonts with cytoplasmic clefts were not common in our material due to the fixatives used (Bouin's and Carnoy's). Merozoites were occasionally observed inside monocytes, probably being phagocytosed.     

Purification and properties of a triacylglycerol lipase from Mycobacterium phlei.

In order to study the metabolism of triacylglycerol in mycobacteria, an intracellular particulate triacylglycerol lipase (EC 3.1.1.3) was purified 800-fold from stationary phase cells of Mycobacterium phlei. Extraction of whole cell suspensions with 5% Triton X-100, followed by ion-exchange chromatography of the extract on two successive DEAE-cellulose columns produced a preparation which was nearly homogeneous by the criterion of analytical isoelectric focusing in acrylamide gels (one band, pI. 3.8) and by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the preparation into six protein bands. Lipase activity stable to electrophoresis in sodium dodecyl sulfate was extracted from the 40 000 molecular weight region of the gels. ith phosphate or maleate buffer the enzyme exhibits a broad pH optimum around 6.0 with sigmoid saturation kinetics (Hill number 2), and an apparent Km of 8.8 mM for tripalmitoylglycerol. Citrate and other carboxylic acids increase the apparent V up to 3-fold with the Hill number approaching 1.0. In a series of p-nitrophenyl esters tested (C2-C18), p-nitrophenylmyristate was hydrolyzed most rapidly. The saturation curve for p=nitrophenylmyristate was sigmoid and unaffected by citrate. The role of this activity in the metabolism of triacylglycerols by Mycobacteria is discussed.