Influence of preovulatory estradiol treatment on the maintenance of pregnancy in beef cattle receiving in vivo produced embryos.

This experiment was designed to determine the role of preovulatory estradiol in pregnancy retention after embryo transfer (ET). Cows were synchronized with the 7-d CO-Synch + CIDR® protocol. On d0 (d-2 =CIDR® removal), cows were grouped by estrual status (estrual [Positive Control] and nonestrual), and nonestrual cows were administered Gonadotropin Releasing Hormone (GnRH) and randomly assigned to either no treatment (Negative Control) or Estradiol (0.1 mg estradiol 17-ß IM). All cows received an embryo on d7. Pregnancy status was retrospectively classified on d56, 30, 24, and 19 by either ultrasonography, plasma pregnancy-associated glycoproteins analysis (PAGs), expression of interferon-stimulated genes, plasma progesterone (P4) concentrations, or a combination of the factors. There was no difference in estradiol concentrations on day 0 h 0 (P > 0.16). At day 0 h 2, Estradiol cows (15.7 ± 0.25 pg/mL) had elevated (P < 0.001) estradiol compared with Positive Controls (3.4 ± 0.26 pg/mL) or Negative Controls (4.3 ± 0.25 pg/mL). On d19, pregnancy rates did not differ (P = 0.14) among treatments. On d24, Positive Controls (47%) had greater (P < 0.01) pregnancy rates than Negative Controls (32%); Estradiol cows were intermediate (40%). There was no difference (P = 0.38) in pregnancy rates between Positive Control (41%) and Estradiol (36%) cows on d30, but Negative Control (27%) cows had (P = 0.01) or tended (P = 0.08) to have decreased pregnancy rates, respectively. Thus, preovulatory estradiol may elicit an effect on early uterine attachment or alter histotroph components, consequently improving pregnancy maintenance through d30.

RNA sequencing and iTRAQ proteomic data from an experiment examining the influence of conceptus presence and preovulatory estradiol on endometrial gene transcripts and proteins around maternal recognition of pregnancy in beef cattle.

RNA sequencing reads and isobaric tags for a relative and absolute quantification (iTRAQ)-Based Proteomic Data were used to determine the impact of conceptus presence and preovulatory estradiol concentration on function of the d16 uterus in beef cattle. Conceptuses and endometrial biopsies were collected from the uterine horn ipsilateral to the corpus luteum. Total cellular RNA was extracted from endometrium for RNA sequencing across two lanes of a NovaSeq S2, 2 × 50-bp run. Two independent uterine luminal fluid pools (ULF) were made for each group: highE2/conceptus, highE2/noconceptus, lowE2/conceptus, and lowE2/noconceptus. Peptides were labeled with iTRAQ reagents and analyzed using 2-dimensional liquid chromatography mass spectrometry. Transcript abundances were determined using DESeq2 (FDR <0.05, FC>2). Scaffold Q+ was used to quantitate peptide and protein identifications in ULF. Datasets include uterine transcript and protein abundances among highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus groups. This information can be useful for further investigating the role of specific transcripts and proteins in the maintenance of early pregnancy in beef cattle. This dataset is related to the article 'Influence of conceptus presence and preovulatory estradiol exposure on uterine gene transcripts and proteins around maternal recognition of pregnancy in beef cattle' by E.J. Northrop-Albrecht, J.J.J. Rich, R.A. Cushman, R. Yao, X. Ge, G.A. Perry. Molecular and Cellular Endocrinology.

Effect of estradiol preceding and progesterone subsequent to ovulation on proportion of postpartum beef cows pregnant.

Two experiments were conducted to examine the effect of plasma concentrations of 17ß-estradiol (E2) preceding and progesterone (P4) subsequent to ovulation on proportions of beef cows pregnant following embryo transfer. Timing of ovulation (d 0) among postpartum cows was synchronized and cows that expressed estrus were removed from each study. In Experiment 1, plasma E2 concentration on d 0 was used to classify cows (n = 353) into Low, Medium, and High E2 groups. Pregnancy rate for cows with Low, Medium, or High E2 concentrations were different (P < 0.05). In Experiment 2, there were multiple administrations of PGF2α to evaluate the independent effects of Low or High E2 before ovulation and Low or Normal (no treatment) P4 after ovulation on proportions of cows pregnant. Treatment groups in Experiment 2, therefore, were: Low E2-Low P4 (LL; n = 71), Low E2-Normal P4 (LN; n = 69), High E2-Low P4 (HL; n = 74), and High E2-Normal P4 (HN; n = 73). Concentrations of P4 on d 7 subsequent to ovulation were less (P < 0.05) in cows of the HL compared to HN, and in LL compared to LN groups. Concentrations of E2 on d -2, 0, and change in E2 (d -2 to d 0) had a positive effect (P < 0.008) on pregnancy rates. In summary, relatively greater E2 concentrations preceding ovulation; and relatively greater P4 concentrations subsequent to ovulation combined with lesser E2 concentrations preceding ovulation had a positive effect on proportions of postpartum cows pregnant.

Denitrification as the dominant nitrogen loss process in the Arabian Sea.

Primary production in over half of the world's oceans is limited by fixed nitrogen availability. The main loss term from the fixed nitrogen inventory is the production of dinitrogen gas (N(2)) by heterotrophic denitrification or the more recently discovered autotrophic process, anaerobic ammonia oxidation (anammox). Oceanic oxygen minimum zones (OMZ) are responsible for about 35% of oceanic N(2) production and up to half of that occurs in the Arabian Sea. Although denitrification was long thought to be the only loss term, it has recently been argued that anammox alone is responsible for fixed nitrogen loss in the OMZs. Here we measure denitrification and anammox rates and quantify the abundance of denitrifying and anammox bacteria in the OMZ regions of the Eastern Tropical South Pacific and the Arabian Sea. We find that denitrification rather than anammox dominates the N(2) loss term in the Arabian Sea, the largest and most intense OMZ in the world ocean. In seven of eight experiments in the Arabian Sea denitrification is responsible for 87-99% of the total N(2) production. The dominance of denitrification is reproducible using two independent isotope incubation methods. In contrast, anammox is dominant in the Eastern Tropical South Pacific OMZ, as detected using one of the isotope incubation methods, as previously reported. The abundance of denitrifying bacteria always exceeded that of anammox bacteria by up to 7- and 19-fold in the Eastern Tropical South Pacific and Arabian Sea, respectively. Geographic and temporal variability in carbon supply may be responsible for the different contributions of denitrification and anammox in these two OMZs. The large contribution of denitrification to N(2) loss in the Arabian Sea indicates the global significance of denitrification to the oceanic nitrogen budget.

Responses of nitrification and ammonia-oxidizing bacteria to reciprocal transfers of soil between adjacent coniferous forest and meadow vegetation in the Cascade Mountains of Oregon.

Despite the critical position of nitrification in N cycling in coniferous forest soils of western North America, little information exists on the composition of ammonia-oxidizing bacteria (AOB) in these soils, or their response to treatments that promote or reduce nitrification. To this end, an experiment was conducted in which a set of soil cores was reciprocally transplanted between adjacent forest (low nitrification potential) and meadow (high nitrification potential) environments, at two high-elevation (approximately 1500 m) sites in the H.J. Andrews Experimental Forest located in the Cascade Mountains of Oregon. Half of the cores were placed in screened PVC pipe (closed) to prevent new root colonization, large litter debris inputs, and animal disturbance; the other cores were placed in open mesh bags. A duplicate set of open and closed soil cores was not transferred between sites and was incubated in place. Over the 2-year experiment, net nitrification increased in both open and closed cores transferred from forest to meadow, and to a lesser extent in cores remaining in the forest. In three of four forest soil treatments, net nitrification increases were accompanied by increases in nitrification potential rates (NPR) and 10- to 100-fold increases in AOB populations. In open cores remaining in the forests, however, increases in net nitrification were not accompanied by significant increases in either NPR or AOB populations. Although some meadow soil treatments reduced both net nitrification and nitrification potential rates, significant changes were not detected in most probable number (MPN)-based estimates of AOB population densities. Terminal restriction fragment profiles (T-RFs) of a PCR-amplified 491-bp fragment of the ammonia monooxygenase subunit A gene (amoA) changed significantly in response to some soil treatments, and treatment effects differed among locations and between years. A T-RF previously shown to be a specific biomarker of Nitrosospira cluster 4 (Alu390) was widespread and dominant in the majority of soil samples. Despite some treatments causing substantial increases in AOB population densities and nitrification potential rates, nitrosomonads remained undetectable, and the nitrosospirad AOB community composition did not change radically following treatment.

Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils.

We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers. nosZ, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of nosZ terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225 nosZ clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of nosZ fragments sequenced from meadow or forest soils were most similar to nosZ from the Rhizobiaceae group in alpha-Proteobacteria species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.

Multiple loci of Pseudomonas syringae pv. syringae are involved in pathogenicity on bean: restoration of one lesion-deficient mutant requires two tRNA genes.

A mutational analysis of lesion-forming ability was undertaken in Pseudomonas syringae pv. syringae B728a, causal agent of bacterial brown spot disease of bean. Following a screen of 6,401 Tn5-containing derivatives of B728a on bean pods, 26 strains that did not form disease lesions were identified. Nine of the mutant strains were defective in the ability to elicit the hypersensitive reaction (HR) and were shown to contain Tn5 insertions within the P. syringae pv. syringae hrp region. Ten HR+ mutants were defective in the production of the toxin syringomycin, and a region of the chromosome implicated in the biosynthesis of syringomycin was deleted in a subset of these mutants. The remaining seven lesion-defective mutants retained the ability to produce protease and syringomycin. Marker exchange mutagenesis confirmed that the Tn5 insertion was causal to the mutant phenotype in several lesion-defective, HR+ strains. KW239, a lesion- and syringomycin-deficient mutant, was characterized at the molecular level. Sequence analysis of the chromosomal region flanking the Tn5 within KW239 revealed strong similarities to a number of known Escherichia coli gene products and DNA sequences: the nusA operon, including the complete initiator tRNA(Met) gene, metY; a tRNA(Leu) gene; the tpiA gene product; and the MrsA protein. Removal of sequences containing the two potential tRNA genes prevented restoration of mutant KW239 in trans. The Tn5 insertions within the lesion-deficient strains examined, including KW239, were not closely linked to each other or to the lemA or gacA genes previously identified as involved in lesion formation by P. syringae pv. syringae.

Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae.

Mutational analysis of the bean-pathogenic Pseudomonas syringae pv. syringae strain B728a has led to the genetic identification of the gacA gene as encoding the response regulator for the unlinked lemA sensor kinase. The analysis of a collection of spontaneous mutants of P. syringae pv. syringae suggested that the gacA gene was involved in lesion formation and the production of protease and syringomycin. The gacA gene originally was identified as a regulator of extracellular antibiotic production by Pseudomonas fluorescens, and the predicted GacA protein is a member of the FixJ family of bacterial response regulators. The sequence of the putative B728a GacA protein revealed 92% identity with the P. fluorescens GacA protein. An insertional mutation within the P. syringae pv. syringae gacA gene abrogated lesion formation on beans, production of extracellular protease, and production of the toxin syringomycin, the same phenotypes affected by a lemA mutation. DNA sequence analysis identified the P. syringae pv. syringae uvrC gene immediately downstream of the gacA gene, an arrangement conserved in P. fluorescens and Escherichia coli. The gacA insertional mutant was sensitive to UV, presumably because of polarity on transcription of the downstream uvrC gene. Southwestern (DNA-protein) analysis revealed that the lemA and gacA genes were required for the full expression of a DNA binding activity.

Pathovar-specific requirement for the Pseudomonas syringae lemA gene in disease lesion formation.

The lemA gene is conserved among strains and pathovars of Pseudomonas syringae. In P. syringae pv. syringae B728a, a causal agent of bacterial brown spot disese of bean, the lemA gene is required for lesion formation on leaves and pods. Using lemA-containing DNA as a probe, we determined that 80 P. syringae pv. syringae strains isolated from bean leaves could be grouped into seven classes based on restriction fragment length polymorphism. Marker exchange mutagenesis showed that the lemA gene was required for lesion formation by representative strains from each restriction fragment length polymorphism class. Hybridization to the lemA locus was detected within six different P. syringae pathovars and within Pseudomonas aeruginosa. Interestingly, a lemA homolog was present and functional within the nonpathogenic strain P. syringae Cit7. We cloned a lemA homolog from a genomic library of P. syringae pv. phaseolicola NPS3121, a causal agent of halo blight of bean, that restored lesion formation to a P. syringae pv. syringae lemA mutant. However, a lemA mutant P. syringae pv. phaseolicola strain retained the ability to produce halo blight disease symptoms on bean plants. Therefore, the lemA gene played an essential role in disease lesion formation by P. syringae pv. syringae isolates, but was not required for pathogenicity of a P. syringae pv. phaseolicola strain.

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.