Structure and Mechanisms of Assembly-Line Polyketide Synthases.

Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS. Looking forward, one anticipates major advances in our understanding of the mechanisms by which the free energy of a repetitive Claisen-like reaction is harnessed to guide the growing polyketide chain along the assembly line in a manner that is kinetically robust yet evolutionarily adaptable.

Structure and mechanism for iterative amide N-methylation in the biosynthesis of channel-forming peptide cytotoxins.

SignificanceThe channel-forming proteusins are bacterial helical peptides that allow permeation of positively charged ions to influence membrane potential and cellular physiology. We biochemically characterize the effect of two critical posttranslational modifications on the secondary structure of the peptide substrate. We determine how a methyl group can be added to the side chains of D-Asn residues in a peptide substrate and show how flanking residues influence selectivity. These studies should foster the development of small-molecule peptide ion channels as therapeutics.

Biological Demands and Toxicity of Isoprenoid Precursors in Bacillus Subtilis Through Cell-Permeant Analogs of Isopentenyl Pyrophosphate and Dimethylallyl Pyrophosphate.

Bacterial isoprenoids are necessary for many biological processes, including maintaining membrane integrity, facilitating intercellular communication, and preventing oxidative damage. All bacterial isoprenoids are biosynthesized from two five carbon structural isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are cell impermeant. Herein, we demonstrate exogenous delivery of IPP and DMAPP into Bacillus subtilis by utilizing a self-immolative ester (SIE)-caging approach. We initially evaluated native B. subtilis esterase activity, which revealed a preference for short straight chain esters. We then examined the viability of the SIE-caging approach in B. subtilis and demonstrate that the released caging groups are well tolerated and the released IPP and DMAPP are bioavailable, such that isoprenoid biosynthesis can be rescued in the presence of pathway inhibitors. We further show that IPP and DMAPP are both toxic and inhibit growth of B. subtilis at the same concentration. Lastly, we establish the optimal ratio of IPP to DMAPP (5 : 1) for B. subtilis growth and find that, surprisingly, DMAPP alone is insufficient to rescue isoprenoid biosynthesis under high concentrations of fosmidomycin. These findings showcase the potential of the SIE-caging approach in B. subtilis and promise to both aid in novel isoprenoid discovery and to inform metabolic engineering efforts in bacteria.

The association between selective dry cow therapy, milking routine and dry cow management practices with somatic cell count in early lactation cows from 21 commercial grazing dairy herds.

This observational study aimed to explore the association of farmer-driven selective dry cow therapy (DCT), milking routine and dry cow management practices with somatic cell count (SCC) in early lactation cows from 21 commercial dairy herds. Milking routine practices evaluated referred to cow preparation for milking, in-lactation mastitis management and recording. Dry cow management practices related to dry cow environment and cleaning, dry-off procedure, milk cessation strategy and calving environment. Data from 2,016 multiparous cows in 21 commercial spring-calving grazing dairy herds were available for the study. Herd owners self-reported DCT (the assignment and administration of DCT was at the discretion of the herd owners with no involvement from the research team), management practices during milking and the dry period. Cow-level last test-day SCC records in 2020 [range = 105 to 285 d in milk (DIM)] and first test-day records in 2021 (range = 5 to 60 DIM) were obtained from milk recording databases. Quarter-level milk sampling was carried out on all cows in late lactation of 2020 (range = 240 to 261 DIM) for bacterial culturing. Bacteriological results were used to define cows with intramammary infection (IMI) when ≥ 1 quarter sample resulted in bacterial growth and there were no contaminated samples from that cow. Mixed model analyses were used to evaluate the association of selective DCT, milking routine and dry cow management practices with cows' first test-day log 10 SCC (logSCC) in 2021. At dry-off in 2020, 47.6% of the cows were administered an internal teat sealant alone (ITS) while 52.4% were administered an antibiotic plus an internal teat sealant (AB+ITS). The mean herd-level percentage of cows with IMI was 19.7% (range = 9.8% to 39.5%); Staphylococcus aureus accounted for the majority of cow-level IMI (89.9%, 357/397). Between herds, the proportion of cows administered ITS ranged from 17.7% (14/79; in a herd with an IMI prevalence of 10.1%) to 86.8% (66/76; in a herd with an IMI prevalence of 27.6%). In total, 11.8% (105/889) and 29.8% (292/980) of cows that were administered ITS or AB+ITS had an IMI in late lactation 2020, respectively. The mean untransformed SCC at the last test-day in 2020 of cows administered ITS and AB+ITS was 55,000 and 197,200 cells/mL, respectively. The statistical analysis showed a significant interaction between selective DCT and milk yield at last test-day in 2020; cows with a milk yield of 15 kg and administered ITS had a 0.1 higher (untransformed SCC of 19,000 cells/mL higher) first test-day logSCC compared with cows administered AB+ITS. Additionally, greater parity, IMI in late lactation, higher log SCC at the last test-day in 2020 and longer dry periods were associated with higher logSCC at the first test-day in 2021. The current study identified cow- and herd-level management practices that could aid dairy farmers in improving the outcome of selective DCT and decrease early lactation SCC.

Discovery and Characterization of Antibody Probes of Module 2 of the 6-Deoxyerythronolide B Synthase.

Fragment antigen-binding domains of antibodies (Fabs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of Fabs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two Fabs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediated reactions catalyzed by this module, albeit by distinct mechanisms. AC2 primarily affects the rate (kcat), whereas BB1 increases the KM of an ACP-mediated reaction. A third Fab, AA5, binds to the KS-AT fragment of DEBS Module 2 without altering either parameter; it is phenotypically reminiscent of a previously characterized Fab, 1B2, shown to principally recognize the N-terminal helical docking domain of DEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-AT fragment of Module 2 were solved to 2.70 and 2.65 Å resolution, respectively, and revealed entirely distinct recognition features of the two antibodies. The new tools and insights reported here pave the way toward advancing our understanding of the structure-function relationships of DEBS Module 2, arguably the most well-studied module of an assembly line PKS.

Membrane Permeant Analogs for Independent Cellular Introduction of the Terpene Precursors Isopentenyl- and Dimethylallyl-Pyrophosphate.

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the ß-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP.

Evaluation of test-day milk somatic cell count to predict intramammary infection in late lactation grazing dairy cows.

Use of selective dry cow antimicrobial therapy requires to precisely differentiate cows with an intramammary infection (IMI) from uninfected cows close to drying-off to enable treatment allocation. Milk somatic cell count (SCC) is an indicator of an inflammatory response in the mammary gland and is usually associated with IMI. However, SCC can also be influenced by cow-level variables such as milk yield, lactation number and stage of lactation. In recent years, predictive algorithms have been developed to differentiate cows with IMI from cows without IMI based on SCC data. The objective of this observational study was to explore the association between SCC and subclinical IMI, taking cognizance of cow-level predictors on Irish seasonal spring calving, pasture-based systems. Additionally, the optimal test-day SCC cut-point (maximized sensitivity and specificity) for IMI diagnosis was determined. A total of 2,074 cows, across 21 spring calving dairy herds with an average monthly milk weighted bulk tank SCC of ≤200,000 cells/mL were enrolled in the study. Quarter-level milk sampling was carried out on all cows in late lactation (interquartile range = 240-261 d in milk) for bacteriological culturing. Bacteriological results were used to define cows with IMI, when ≥1 quarter sample resulted in bacterial growth. Cow-level test-day SCC records were provided by the herd owners. The ability of the average, maximum and last test-day SCC to predict infection were compared using receiver operator curves. Predictive logistic regression models tested included parity (primiparous or multiparous), yield at last test-day and a standardized count of high SCC test-days. In total, 18.7% of cows were classified as having an IMI, with first parity cows having a higher proportion of IMI (29.3%) compared with multiparous cows (16.1%). Staphylococcus aureus accounted for the majority of these infections. The last test-day SCC was the best predictor of infection with the highest area under the curve. The inclusions of parity, yield at last test-day, and a standardized count of high SCC test-days as predictors did not significantly improve the ability of last test-day SCC to predict IMI. The cut-point for last test-day SCC which maximized sensitivity and specificity was 64,975 cells/mL. This study indicates that in Irish seasonal pasture-based dairy herds, with low bulk tank SCC control programs, the last test-day SCC (interquartile range days in milk = 221-240) is the best predictor of IMI in late lactation.

Reactive Oxygen Species (ROS) Activated Liposomal Cell Delivery using a Boronate-Caged Guanidine Lipid.

We report boronate-caged guanidine-lipid 1 that activates liposomes for cellular delivery only upon uncaging of this compound by reactive oxygen species (ROS) to produce cationic lipid products. These liposomes are designed to mimic the exceptional cell delivery properties of cell-penetrating peptides (CPPs), while the inclusion of the boronate cage is designed to enhance selectivity such that cell entry will only be activated in the presence of ROS. Boronate uncaging by hydrogen peroxide was verified by mass spectrometry and zeta potential (ZP) measurements. A microplate-based fluorescence assay was developed to study the ROS-mediated vesicle interactions between 1-liposomes and anionic membranes, which were further elucidated via dynamic light scattering (DLS) analysis. Cellular delivery studies utilizing fluorescence microscopy demonstrated significant enhancements in cellular delivery only when 1-liposomes were incubated with hydrogen peroxide. Our results showcase that lipid 1 exhibits strong potential as an ROS-responsive liposomal platform for targeted drug delivery applications.

Cyclic Disulfide Liposomes for Membrane Functionalization and Cellular Delivery.

Liposomes are effective therapeutic delivery nanocarriers due to their ability to encapsulate and enhance the pharmacokinetic properties of a wide range of therapeutics. Two primary areas in which improvement is needed for liposomal drug delivery is to enhance the ability to infiltrate cells and to facilitate derivatization of the liposome surface. Herein, we report a liposome platform incorporating a cyclic disulfide lipid (CDL) for the dual purpose of enhancing cell entry and functionalizing the liposome membrane through thiol-disulfide exchange. In order to accomplish this, CDL-1 and CDL-2, composed of lipoic acid (LA) or asparagusic acid (AA) appended to a lipid scaffold, were designed and synthesized. A fluorescence-based microplate immobilization assay was implemented to show that these compounds enable convenient membrane decoration through reaction with thiol-functionalized small molecules. Additionally, fluorescence microscopy experiments indicated dramatic enhancements in cellular delivery when CDLs were incorporated within liposomes. These results demonstrate that multifunctional CDLs serve as an exciting liposome system for surface decoration and enhanced cellular delivery.

Characterization of glutamyl-tRNA-dependent dehydratases using nonreactive substrate mimics.

The peptide natural product nisin has been used as a food preservative for 6 decades with minimal development of resistance. Nisin contains the unusual amino acids dehydroalanine and dehydrobutyrine, which are posttranslationally installed by class I lanthipeptide dehydratases (LanBs) on a linear peptide substrate through an unusual glutamyl-tRNA-dependent dehydration of Ser and Thr. To date, little is known about how LanBs catalyze the transfer of glutamate from charged tRNAGlu to the peptide substrate, or how they carry out the subsequent elimination of the peptide-glutamyl adducts to afford dehydro amino acids. Here, we describe the synthesis of inert analogs that mimic substrate glutamyl-tRNAGlu and the glutamylated peptide intermediate, and determine the crystal structures of 2 LanBs in complex with each of these compounds. Mutational studies were used to characterize the function of the glutamylation and glutamate elimination active-site residues identified through the structural analysis. These combined studies provide insights into the mechanisms of substrate recognition, glutamylation, and glutamate elimination by LanBs to effect a net dehydration reaction of Ser and Thr.

The effect of concentrate supplement type on milk production, nutrient intake, and total-tract nutrient digestion in mid-lactation, spring-calving dairy cows grazing perennial ryegrass (Lolium perenne L.) pasture.

The objective of this study was to evaluate the effect of concentrate supplement type on milk production, nutrient intake, and total-tract nutrient digestion in lactating dairy cows grazing mid-season perennial ryegrass (Lolium perenne L.; PRG) pasture. Twelve primiparous (mean ± standard deviation; 95 ± 30 d in milk and 470 ± 43 kg of body weight) and 68 multiparous (99 ± 24 d in milk and 527 ± 64 kg of body weight) lactating dairy cows were blocked based on pre-study milk yield and parity and randomly assigned to 1 of 4 dietary treatments. The 4 dietary treatments were a non-supplemented PRG control (PRG); PRG supplemented with 4.4 kg of dry matter (DM) per cow per day of citrus pulp and 0.067 kg of DM/cow per day of urea (PRG+C); PRG supplemented with 0.8 kg of DM/cow per day of heat-treated soybean meal (PRG+PP); and PRG supplemented with 3.1 kg of DM/cow per day of a combination of heat-treated soybean meal and citrus pulp (PRG+C+PP). The study consisted of a 2-wk adaptation period and a 10-wk period of data collection. Weekly measurements of milk yield, body weight, body condition score, and feeding and rumination time were made. Nutrient intake and total-tract digestibility were measured during wk 6 of the study. A large soil moisture deficit was experienced during the study that probably reduced herbage growth rate and likely altered the chemical composition of the PRG offered when compared with typical mid-season PRG. Total dry matter intake was increased in cows fed PRG+C compared with cows fed PRG and PRG+PP and was similar to cows fed PRG+C+PP (18.0, 15.9, 16.4, and 17.2 ± 0.41 kg of DM/d, respectively). The apparent total-tract neutral detergent fiber digestibility of cows fed the PRG+C diet was lower compared with the PRG and PRG+PP diets and was similar to the PRG+C+PP diet (0.67, 0.70, 0.70, and 0.69 ± 0.01 g/g, respectively). The energy-corrected milk (ECM) yield of cows fed PRG+C+PP was highest (23.7 kg/d), PRG+C was intermediate (22.2 kg/d), and PRG was lowest (20.8 kg/d). Cows fed PRG+PP produced more ECM (22.9 kg/d) compared with cows fed PRG and produced similar ECM compared with cows fed PRG+C and PRG+C+PP diets. The PRG+PP diet increased milk protein yield compared with the PRG diet, tended to increase milk protein yield compared with the PRG+C diet, and was similar to the PRG+C+PP diet. Milk fat concentration and the composition of milk fat were not influenced by treatment. The results demonstrated that, for cows consuming pasture-based diets, increasing metabolizable protein supply allowed higher milk yield as metabolizable protein was more limiting than metabolizable energy. However, due to the large soil moisture deficit experienced during this experiment, caution is recommended when extrapolating these results to cows consuming typical mid-season PRG herbage.

Microbial composition and omasal flows of bacterial, protozoal, and nonmicrobial amino acids in lactating dairy cows fed fresh perennial ryegrass (Lolium perenne L.) not supplemented or supplemented with rolled barley.

The objective of this study was to evaluate the effect of rolled barley supplementation on microbial composition and omasal flows of bacterial, protozoal, and nonmicrobial AA in cows fed fresh perennial ryegrass (Lolium perenne L.; PRG). Ten ruminally cannulated multiparous Holstein cows averaging (mean ± standard deviation) 49 ± 23 d in milk and 513 ± 36 kg of body weight were assigned to 1 of 2 treatments in a switchback design. The treatment diets were PRG only or PRG plus 3.5 kg of dry matter rolled barley (G+RB). The study consisted of three 29-d periods where each period consisted of 21 d of diet adaptation and 8 d of data and sample collection. A double-marker system was used to quantify nutrient flow entering the omasal canal along with 15N-ammonium sulfate to label and measure the microbial and nonmicrobial omasal flow of AA. Overall, rolled barley supplementation had no effect on the AA composition of the omasal liquid-associated and particle-associated bacteria. Rolled barley supplementation affected the AA concentrations of omasal protozoa; however, the differences were nutritionally minor. Particle-associated bacteria AA flow was increased for all AA, except for Trp and Pro, in cows fed the G+RB diet. Rolled barley supplementation had no effect on protozoal AA flow. On average, protozoa accounted for 23% of the microbial essential AA flow, which ranged from 17 to 28% for Trp and Lys, respectively. The flow of all AA in omasal true digesta increased in cows fed the G+RB diet compared with the PRG-only diet, resulting in a 228 g/d increase in total AA flow in cows fed the G+RB diet. This increase in total AA flow in cows fed the G+RB diet was due to an increase in microbial AA flow. Rolled barley supplementation had no effect on nonmicrobial AA flow. The nonmicrobial AA flow modestly contributed to total AA flow, accounting for 15.6% on average. These results indicated that extensive ruminal degradation of PRG AA occurred (83.5%), and we demonstrated that cows consuming PRG-based diets exhibit a large dependence on microbial AA to support metabolizable AA supply. Rolled barley supplementation can increase the omasal flow of microbial AA in cows consuming PRG-based diets. However, further research is required to elucidate if this increased AA supply can support higher milk yield under such dietary conditions.

Antibody Probes of Module 1 of the 6-Deoxyerythronolide B Synthase Reveal an Extended Conformation During Ketoreduction.

The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase (PKS) that synthesizes the macrocyclic core of the antibiotic erythromycin. Each of its six multidomain modules presumably sample distinct conformations, as biosynthetic intermediates tethered to their acyl carrier proteins interact with multiple active sites during the courses of their catalytic cycles. The spatiotemporal details underlying these protein dynamics remain elusive. Here, we investigate one aspect of this conformational flexibility using two domain-specific monoclonal antibody fragments (Fabs) isolated from a very large naïve human antibody library. Both Fabs, designated 1D10 and 2G10, were bound specifically and with high affinity to the ketoreductase domain of DEBS module 1 (KR1). Comparative kinetic analysis of stand-alone KR1 as well as a truncated bimodular derivative of DEBS revealed that 1D10 inhibited KR1 activity whereas 2G10 did not. Co-crystal structures of each KR1-Fab complex provided a mechanistic rationale for this difference. A hybrid PKS module harboring KR1 was engineered, whose individual catalytic domains have been crystallographically characterized at high resolution. Size exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS) of this hybrid module bound to 1D10 provided further support for the catalytic relevance of the "extended" model of a PKS module. Our findings reinforce the power of monoclonal antibodies as tools to interrogate structure-function relationships of assembly line PKSs.

Substrate-assisted enzymatic formation of lysinoalanine in duramycin.

Duramycin is a heavily post-translationally modified peptide that binds phosphatidylethanolamine. It has been investigated as an antibiotic, an inhibitor of viral entry, a therapeutic for cystic fibrosis, and a tumor and vasculature imaging agent. Duramycin contains a ß-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) cross-link. The mechanism of Lal formation is not known. Here we show that Lal is installed stereospecifically by DurN via addition of Lys19 to a dehydroalanine. The structure of DurN reveals an unusual dimer with a new fold. Surprisingly, in the structure of duramycin bound to DurN, no residues of the enzyme are near the Lal cross-link. Instead, Hya15 of the substrate makes interactions with Lal, suggesting it acts as a base to deprotonate Lys19 during catalysis. Biochemical data suggest that DurN preorganizes the reactive conformation of the substrate, such that the Hya15 of the substrate can serve as the catalytic base for Lal formation.

Structural insights into enzymatic [4+2] aza-cycloaddition in thiopeptide antibiotic biosynthesis.

The [4+2] cycloaddition reaction is an enabling transformation in modern synthetic organic chemistry, but there are only limited examples of dedicated natural enzymes that can catalyze this transformation. Thiopeptides (or more formally thiazolyl peptides) are a class of thiazole-containing, highly modified, macrocyclic secondary metabolites made from ribosomally synthesized precursor peptides. The characteristic feature of these natural products is a six-membered nitrogenous heterocycle that is assembled via a formal [4+2] cycloaddition between two dehydroalanine (Dha) residues. This heteroannulation is entirely contingent on enzyme activity, although the mechanism of the requisite pyridine/dehydropiperidine synthase remains to be elucidated. The unusual aza-cylic product is distinct from the more common carbocyclic products of synthetic and biosynthetic [4+2] cycloaddition reactions. To elucidate the mechanism of cycloaddition, we have determined atomic resolution structures of the pyridine synthases involved in the biosynthesis of the thiopeptides thiomuracin (TbtD) and GE2270A (PbtD), in complex with substrates and product analogs. Structure-guided biochemical, mutational, computational, and binding studies elucidate active-site features that explain how orthologs can generate rigid macrocyclic scaffolds of different sizes. Notably, the pyridine synthases show structural similarity to the elimination domain of lanthipeptide dehydratases, wherein insertions of secondary structural elements result in the formation of a distinct active site that catalyzes different chemistry. Comparative analysis identifies other catalysts that contain a shared core protein fold but whose active sites are located in entirely different regions, illustrating a principle predicted from efforts in de novo protein design.

Rumen metabolism, omasal flow of nutrients, and microbial dynamics in lactating dairy cows fed fresh perennial ryegrass (Lolium perenne L.) not supplemented or supplemented with rolled barley grain.

The objective of this study was to evaluate the effect of rolled barley grain (RB) supplementation on rumen metabolism, omasal flow of nutrients, and microbial dynamics in lactating dairy cows fed fresh perennial ryegrass (Lolium perenne L.; PRG)-based diets. Ten ruminally cannulated Holstein cows averaging (mean ± standard deviation) 49 ± 23 d in milk and 513 ± 36 kg of body weight were assigned to 1 of 2 treatments in a switchback design. The treatment diets were PRG only (G) or PRG plus 3.5 kg of dry matter RB (G+RB). The study consisted of three 29-d periods where each period consisted of 21 d of diet adaptation and 8 d of data and sample collection. A double marker system was used to quantify nutrient flow entering the omasal canal along with labeled 15N-ammonium sulfate to measure bacterial, protozoal, and nonmicrobial N flow. Rumen evacuation techniques were used to determine nutrient and microbial pool size, allowing the calculation of fractional rates of digestion and microbial growth. There was no difference in daily milk yield or energy-corrected milk yield between treatments. Milk fat concentration and milk urea N decreased, whereas milk protein concentration increased in cows fed the G+RB diet. During the omasal sampling phase, dry matter intake was higher in cows fed the G+RB diet. Ruminal and total-tract neutral detergent fiber digestibility was lower in G+RB cows; however, no difference was observed in reticulorumen pH. The rumen pool size of fermentable carbohydrate was increased in cows fed the G+RB diet; however, the fractional rate of digestion was decreased. Flow of nonammonia N and bacterial N at the omasal canal increased in cows fed the G+RB diet compared with the G diet. Protozoa N flow was not different between diets; however, protozoa appeared to supply a much larger amount of microbial N and exhibited shorter generation time than previously considered. Feed N ruminal digestibility, corrected for microbial contribution, was similar for both treatments (88.4 and 89.0% for G and G+RB, respectively). In conclusion, RB supplementation did not benefit overall animal performance; however, it reduced ruminal neutral detergent fiber digestibility and increased bacterial N flow. The results demonstrate the large dependence of cows consuming PRG-based diets on microbial N as the main source of nonammonia N supply. Additional quantitative research is required to further describe the supply of nutrients and microbial dynamics in cows consuming PRG-based diets in an effort to determine most limiting nutrients.

Structural basis of transcriptional regulation by CouR, a repressor of coumarate catabolism, in Rhodopseudomonas palustris.

The MarR family transcriptional regulator CouR, from the soil bacterium Rhodopseudomonas palustris CGA009, has recently been shown to negatively regulate a p-coumarate catabolic operon. Unlike most characterized MarR repressors that respond to small metabolites at concentrations in the millimolar range, repression by CouR is alleviated by the 800-Da ligand p-coumaroyl-CoA with high affinity and specificity. Here we report the crystal structures of ligand-free CouR as well as the complex with p-coumaroyl-CoA, each to 2.1-Å resolution, and the 2.85-Å resolution cocrystal structure of CouR bound to an oligonucleotide bearing the cognate DNA operator sequence. In combination with binding experiments that uncover specific residues important for ligand and DNA recognition, these structures provide glimpses of a MarR family repressor in all possible states, providing an understanding of the molecular basis of DNA binding and the conformation alterations that accompany ligand-induced dissociation for activation of the operon.

Effect of using internal teat sealant with or without antibiotic therapy at dry-off on subsequent somatic cell count and milk production.

The objective of this study was to assess the effect of treating cows with teat sealant only compared with antibiotic plus teat sealant at drying off on weekly somatic cell count, potential intramammary infection, and milk production across the entire subsequent lactation. In 3 research herds in the south of Ireland, cows with SCC that did not exceed 200,000 cells/mL in the previous lactation (LowSCC) were randomly assigned to 1 of 2 treatments at drying off: internal teat sealant alone (ITS) or antibiotic plus teat sealant (AB+ITS). Cows with SCC that exceeded 200,000 cells/mL in the previous lactation were treated with AB+ITS and included in the analyses as a separate group (HighSCC). Weekly individual animal composite SCC records were available for 654 cow lactations and were transformed to somatic cell scores (SCS) for the purpose of analysis. Data were divided into 3 data sets to represent records obtained (1) up to 35 DIM, (2) up to 120 DIM, and (3) across the lactation. Foremilk secretions were taken from all quarters at drying off, at calving, 2 wk after calving, and in mid-lactation and were cultured to detect the presence of bacteria. The LowSCC cows treated with ITS alone had higher daily milk yield (0.67 kg/d) across lactation compared with LowSCC cows treated with AB+ITS. The LowSCC cows treated with ITS alone had higher SCS in early, up to mid, and across lactation compared with LowSCC cows treated with AB+ITS. We detected no difference in weekly SCS of LowSCC cows treated with ITS alone and SCS of HighSCC cows. The least squares means back-transformed SCC across lactation of the LowSCC cows treated with ITS alone, LowSCC cows treated with AB+ITS, and HighSCC cows were 41,523, 34,001, and 38,939 cells/mL respectively. The odds of LowSCC cows treated with ITS alone having bacteria present in their foremilk across lactation was 2.7 (95% confidence interval: 1.91 to 3.85) and 1.6 (1.22 to 2.03) times the odds of LowSCC cows treated with AB+ITS and of HighSCC cows treated with AB+ITS, respectively. In this study, Staphylococcus aureus was the most prevalent pathogen isolated from the population. Recategorizing the threshold for LowSCC cows as ≤150,000 cells/mL or ≤100,000 cells/mL in the previous lactation had no effect on the results. The results indicate that herds with good mastitis control programs may use ITS alone at dry-off in cows with SCC <200,000 cells/mL across lactation with only a small effect on herd SCC.

Intake, efficiency, and feeding behavior characteristics of Holstein-Friesian cows of divergent Economic Breeding Index evaluated under contrasting pasture-based feeding treatments.

The objective of the current study was to explore differences in dry matter intake, intake capacity, production efficiency, energy balance, and grazing behavior, of 2 divergent genetic groups (GG) of lactating Holstein-Friesian, selected using the Irish Economic Breeding Index (EBI). The GG were evaluated across 3 spring calving pasture-based feeding treatments (FT) over 3 yr. The 2 divergent GG were (1) high EBI, representative of the top 5% nationally (elite), and (2) EBI representative of the national average (NA). In each year 90 elite and 45 NA cows were randomly allocated to 1 of 3 FT: control, lower grass allowance, and high concentrate. Although FT did affect animal performance, there were few notable incidences of GG × FT interaction. The elite cows expressed lower daily milk yield (-1 kg) compared with NA. Elite cows did, however, express higher daily concentrations of milk fat (+3.7 g/kg) and protein (+2.1 g/kg) compared with NA. Daily yield of milk solids and net energy of lactation (NEL) was similar for both GG. Body weight (BW) was greater for NA (+13 kg) compared with elite, whereas mean body condition score was greater (+0.14) for elite compared with NA. Intake did not differ significantly between GG. Intake capacity, expressed as total dry matter intake/100 kg of BW, was greater with elite compared with NA. Production efficiency expressed as yield of milk solids per 100 kg of BW was greater with elite compared with NA, although milk solids/total dry matter intake did not differ between GG. Expressed as NEL as a proportion of net energy intake minus net energy of maintenance (NEL/NEI - NEM) and NEI/milk solids kg, indicated a slight reduction in the utilization of ingested energy for milk production with elite compared with NA. This is, however, suggested as favorable as it manifested as a more positive energy balance with elite compared with NA and so is likely to enhance robustness, increase longevity, and increase overall lifetime efficiency. Noteworthy was a consistent numerical trend toward more intense grazing activity with elite compared with NA cows, exhibited in the numerically greater grazing time (+19 min) and total number of bites per day (+2,591).

A 100-Year Review: A century of change in temperate grazing dairy systems.

From 1917 to 2017, dairy grazing systems have evolved from uncontrolled grazing of unimproved pastures by dual-purpose dairy-beef breeds to an intensive system with a high output per unit of land from a fit-for-purpose cow. The end of World War I signaled significant government investments in agricultural research institutes around the world, which coincided with technological breakthroughs in milk harvesting and a recognition that important traits in both plants and animals could be improved upon relatively rapidly through genetic selection. Uptake of milk recording and herd testing increased rapidly through the 1920s, as did the recognition that pastures that were rested in between grazing events yielded more in a year than those continuously grazed. This, and the invention and refinement of the electric fence, led to the development of "controlled" rotational grazing. This, in itself, facilitated greater stocking rates and a 5 to 10% increase in milk output per hectare but, perhaps more importantly, it allowed a more efficient use of nitrogen fertilizer, further increasing milk output/land area by 20%. Farmer inventions led to the development of the herringbone and rotary milking parlors, which, along with the "unshortable" electric fence and technological breakthroughs in sperm dilution rates, allowed further dairy farm expansion. Simple but effective technological breakthroughs in reproduction ensured that cows were identified in estrus early (a key factor in maintaining the seasonality of milk production) and enabled researchers to quantify the anestrus problem in grazing herds. Genetic improvement of pasture species has lagged its bovine counterpart, but recent developments in multi-trait indices as well as investment in genetic technologies should significantly increase potential milk production per hectare. Decades of research on the use of feeds other than pasture (i.e., supplementary feeds) have provided consistent milk production responses when the reduction in pasture intake associated with the provision of supplementary feed (i.e., substitution rate) is accounted for. A unique feature of grazing systems research over the last 70 yr has been the use of multi-year farm systems experimentation. These studies have allowed the evaluation of strategic changes to a component of the system on all the interacting features of the system. This technique has allowed excellent component research to be "systemized" and is an essential part of the development of the intensive grazing production system that exists today. Future challenges include the provision of skilled labor or specifically designed automation to optimize farm management and both environmental sustainability and animal welfare concerns, particularly relating to the concentration of nitrogen in each urine patch and the associated risk of nitrate leaching, as well as concerns regarding exposure of animals to harsh climatic conditions. These combined challenges could affect farmers' "social license" to farm in the future.