Microbial contamination of harvested colostrum on Czech dairy farms.

The objective of this study was to perform a quantitative and qualitative evaluation of microbial contamination of harvested colostrum on 39 Czech dairy farms. The study identified the proportion of colostrum samples that met the recommended goals for total plate count (TPC), total coliform count (TCC), and gram-negative noncoliform count (NCC), and evaluated the effect of the farm, breed, parity, season of the year, time of calving, and colostrum volume on these 3 microbiological parameters. Colostrum samples from cows (n = 1,241; 57.6% from Czech Fleckvieh, and 42.4% from Holstein breed) were collected on dairy farms between autumn of 2015 and spring of 2017. The samples were collected after the first milking directly from milking buckets. In 155 out of 1,241 colostrum samples (26 farms, 6 samples each, except 1 farm), the species of microorganisms obtained by culture were determined, and the findings were classified into 4 groups according to the probable source of contamination as follows: (1) normal inhabitants of bovine skin and mucosa, (2) fecal contaminants, (3) environmental contaminants, and (4) potential gram-positive mammary pathogens. Our results showed heavy microbial contamination of collected colostrum samples (TPC median = 408,000 cfu/mL; TCC median = 200 cfu/mL; NCC median = 80 cfu/mL). Only 28.4% of samples met the requirement for TPC (<100,000 cfu/mL), 88.2% for TCC (<10,000 cfu/mL), and 86.0% for NCC (<5,000 cfu/mL). Among the tested factors, we found that farm had a significant effect on all 3 microbiological parameters, volume of colostrum had an effect on TPC (the highest TPC in <3.0 L of colostrum), and season had an effect on TCC and NCC (higher TCC in summer than in autumn and winter; the highest NCC in summer and higher in autumn than in spring and winter). Our results showed that most microbes isolated from colostrum belonged to normal inhabitants of bovine skin and mucosa, fecal, or environmental contaminants (i.e., 82.6%, 81.9%, and 75.5% of colostrum samples, respectively). Potential gram-positive mammary pathogens were found in 13.5% of samples. Escherichia coli was isolated from 9.0% of colostrum samples, and Streptococcus uberis and Streptococcus parauberis were each isolated from 5.2% of samples. Our study showed high microbial contamination of colostrum collected on dairy farms. Therefore, better hygiene and sanitation around colostrum harvest should be addressed by farmers.

Identification of AHK2- and AHK3-like cytokinin receptors in Brassica napus reveals two subfamilies of AHK2 orthologues.

Cytokinin (CK) signalling is known to play key roles in the regulation of plant growth and development, crop yields, and tolerance to both abiotic stress and pathogen defences, but the mechanisms involved are poorly characterized in dicotyledonous crops. Here the identification and functional characterization of sensor histidine kinases homologous to Arabidopsis CK receptors AHK2 and AHK3 in winter oilseed rape are presented. Five CHASE-containing His kinases were identified in Brassica napus var. Tapidor (BnCHK1-BnCHK5) by heterologous hybridization of its genomic library with gene-specific probes from Arabidopsis. The identified bacterial artificial chromosome (BAC) clones were fingerprinted and representative clones in five distinct groups were sequenced. Using a bioinformatic approach and cDNA cloning, the precise gene and putative protein domain structures were determined. Based on phylogenetic analysis, four AHK2 (BnCHK1-BnCHK4) homologues and one AHK3 (BnCHK5) homologue were defined. It is further suggested that BnCHK1 and BnCHK3, and BnCHK5 are orthologues of AHK2 and AHK3, originally from the B. rapa A genome, respectively. BnCHK1, BnCHK3, and BnCHK5 displayed high affinity for trans-zeatin (1-3nM) in a live-cell competitive receptor assay, but not with other plant hormones (indole acetic acid, GA3, and abscisic acid), confirming the prediction that they are genuine CK receptors. It is shown that BnCHK1 and BnCHK3, and BnCHK5 display distinct preferences for various CK bases and metabolites, characteristic of their AHK counterparts, AHK2 and AHK3, respectively. Interestingly, the AHK2 homologues could be divided into two subfamilies (BnCHK1/BnCK2 and BnCHK3/BnCHK4) that differ in putative transmembrane domain topology and CK binding specificity, thus implying potential functional divergence.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana.

Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution.

Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana.

Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²âº, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF3⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²âº. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.

Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid.

Differential distribution of the plant hormone auxin within tissues mediates a variety of developmental processes. Cellular auxin levels are determined by metabolic processes including synthesis, degradation, and (de)conjugation, as well as by auxin transport across the plasma membrane. Whereas transport of free auxins such as naturally occurring indole-3-acetic acid (IAA) is well characterized, little is known about the transport of auxin precursors and metabolites. Here, we identify a mutation in the ABCG37 gene of Arabidopsis that causes the polar auxin transport inhibitor sensitive1 (pis1) phenotype manifested by hypersensitivity to auxinic compounds. ABCG37 encodes the pleiotropic drug resistance transporter that transports a range of synthetic auxinic compounds as well as the endogenous auxin precursor indole-3-butyric acid (IBA), but not free IAA. ABCG37 and its homolog ABCG36 act redundantly at outermost root plasma membranes and, unlike established IAA transporters from the PIN and ABCB families, transport IBA out of the cells. Our findings explore possible novel modes of regulating auxin homeostasis and plant development by means of directional transport of the auxin precursor IBA and presumably also other auxin metabolites.