A novel microbiome metabolic modulator improves the growth performance of broiler chickens in multiple trials and modulates targeted energy and amino acid metabolic pathways in the cecal metagenome.

A meta-analysis of 19 floor-pen trials (579 replicate pen observations) in diverse geographies, basal diets, seasons, and medication programs was carried out to evaluate the effects of 2 precision glycan microbiome metabolic modulators (MMM1 and MMM2) on the performance of broiler chickens. In each trial, negative-control (NC) diets were compared with either MMM1 (14 trials) or MMM2 (8 trials), supplemented at an intended dose of 500 g/MT from hatch to 31 to 42 d. A dose response of MMM2 was evaluated in 8 trials at doses of 100, 250, 500, and 1,000 g/MT, not all present in each trial. Linear mixed-effect models were constructed for the final BW, cumulative feed intake, feed conversion ratio (FCR) corrected by mortality and BW (cFCR), and mortality, with Treatment as the fixed effect, nested random effects of Trial and Block, and adjustments for heterogeneity of variances. A significance level of P < 0.05 was used. In one of the studies, cecal content samples were collected at 42 d for analysis of microbiome gene abundance. Microbiome metabolic modulator 2 exhibited a reduction of the cFCR of 0.06 g feed/g BW gain compared with the NC and 0.03 g feed/g BW gain compared with MMM1, whereas MMM1 reduced the cFCR by 0.03 g feed/g BW gain compared with NC. Both MMM1 and MMM2 increased the final BW compared with the NC by 43 and 48 g/bird, respectively, with no difference among them. Compared with NC, feed intake was increased by MMM1 (+51 g/bird) and reduced by MMM2 (-74 g/bird). A one-directional dose response of the MMM2 ingredient was observed for the final BW (increasing) and cFCR (decreasing), whereas the feed intake response reached a minimum at 500 g/MT. The metagenomic analysis confirmed an increase in the abundance of genes belonging to the acrylate pathway, which is involved in propionate production, as well as arginine-N-succinyl transferase which is involved in the catabolism of arginine, in response to MMM2. Differential glycan structures of the MMM had an impact on the size and consistency of performance effects in broilers.

A region of sigmaK involved in promoter activation by GerE in Bacillus subtilis.

During endospore formation in Bacillus subtilis, the DNA binding protein GerE stimulates transcription from several promoters that are used by RNA polymerase containing sigmaK. GerE binds to a site on one of these promoters, cotX, that overlaps its -35 region. We tested the model that GerE interacts with sigmaK at the cotX promoter by seeking amino acid substitutions in sigmaK that interfered with GerE-dependent activation of the cotX promoter but which did not affect utilization of the sigmaK-dependent, GerE-independent promoter gerE. We identified two amino acid substitutions in sigmaK, E216K and H225Y, that decrease cotX promoter utilization but do not affect gerE promoter activity. Alanine substitutions at these positions had similar effects. We also examined the effects of the E216A and H225Y substitutions in sigmaK on transcription in vitro. We found that these substitutions specifically reduced utilization of the cotX promoter. These and other results suggest that the amino acid residues at positions 216 and 225 are required for GerE-dependent cotX promoter activity, that the histidine at position 225 of sigmaK may interact with GerE at the cotX promoter, and that this interaction may facilitate the initial binding of sigmaK RNA polymerase to the cotX promoter. We also found that the alanine substitutions at positions 216 and 225 of sigmaK had no effect on utilization of the GerE-dependent promoter cotD, which contains GerE binding sites that do not overlap with its -35 region.

A region in the Bacillus subtilis transcription factor Spo0A that is important for spoIIG promoter activation.

Spo0A is a DNA binding protein in Bacillus subtilis required for the activation of spoIIG and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the sigmaA subunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in sigmaA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in sigmaA. We also found that the S231F substitution partially restored use of the spoIIG promoter by the sigmaA H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the spoIIA promoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, sigmaH-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in sigmaA-dependent promoter activation.

Promoter recognition by a cyanobacterial RNA polymerase: in vitro studies with the Calothrix sp. PCC 7601 transcriptional factors RcaA and RcaD.

To study the transcriptional apparatus and the mechanisms that control gene expression in cyanobacteria, the RNA polymerase was purified from the filamentous Calothrix sp. PCC 7601 and used in in vitro transcription assays. Conditions required for specific transcription initiation to occur were analyzed with the eleven Calothrix PCC 7601 genes for which the 5' ends have been mapped. Most of the transcripts directly obtained did not have the expected size, providing a test for looking at specific transcription factors. Addition of RcaA, a protein that binds to the promoter region of the phycobiliprotein cpeBA operon, restored accurate initiation of transcription in the in vitro system for three phycobiliprotein promoters. RcaA thus is a transcription factor that allows to mimick in vivo transcription. In parallel, the functional properties of the Escherichia coli and cyanobacterial RNA polymerases were compared. The enteric enzyme could not precisely initiate transcription at the promoter of a phycobiliprotein gene and, reciprocally, the cyanobacterial RNA polymerase could initiate transcription at PlacUV5, but not from wild-type Plac promoters. The different behaviours of the enzymes are discussed in the light of the structural differences that exist between subunits of the RNA polymerases.

Activation of the Bacillus subtilis spoIIG promoter requires interaction of Spo0A and the sigma subunit of RNA polymerase.

Bacillus subtilis Spo0A activates transcription from both sigmaA- and sigmaH-dependent promoters. Baldus et al. (2) identified two amino acid substitutions in the carboxyl terminus of sigmaA, K356E and H359R, that specifically impaired Spo0A-activated transcription in vivo. To test the model in which the K356E and H359R substitutions in sigmaA interfere with the interaction of Spo0A and sigmaA, we examined the effects of alanine substitutions at these positions in sigmaA on sigmaA's ability to direct transcription in vivo and in vitro. We found that alanine substitutions at these positions specifically reduced expression from the sigmaA-dependent, Spo0A-dependent promoters, spoIIG and spoIIE, in vivo. Furthermore, we found that stimulation of spoIIG promoter activity by Spo0A in vitro was reduced by the single substitutions H359A and H359R in sigmaA.

Prochlorothrix hollandica PCC 9006: genomic properties of an axenic representative of the chlorophyll a/b-containing oxyphotobacteria.

Prochlorothrix hollandica is an oxygenic photosynthetic prokaryote that differs from the cyanobacteria in having chlorophyll a/b-protein complexes instead of phycobilisomes as major light-harvesting antennae. We report the isolation and culturing of an axenic strain of P. hollandica, available from the Pasteur Culture Collection of Cyanobacteria as strain PCC 9006. The strain has a mean DNA base composition of 51.6 +/- 0.1 mol% G+C and a genomic complexity of 3.37 +/- 0.17 x 10(9) daltons (5,505 kb). A reiterated DNA sequence represents approximately 4.4% of the genome. Restriction enzyme isoschizomers with different sensitivities to base methylation were used to demonstrate that most A residues in the sequence GATC are methylated in P. hollandica DNA and that this methylation increases with culture age. Furthermore, some C residues are methylated, although the specificity of the C methylation system does not match that of well-characterized C methylases. Nucleotide analysis showed that up to approximately 3.5% of both dA and dC residues are methylated in P. hollandica DNA.

Specific initiation of transcription at a cyanobacterial promoter with RNA polymerase purified from Calothrix sp. PCC 7601.

Although in cyanobacteria many genes have been shown to be transcriptionally controlled by specific stimuli, little is known about promoter structure and the form of RNA polymerase that recognizes individual promoters. RNA polymerase holoenzyme has been purified from Calothrix sp. PCC 7601. Its polypeptide composition resembles that of the plant chloroplast enzymes. To study transcription in cyanobacteria further, we have analysed the promoter-recognition properties of the purified enzyme. In vitro transcription was assayed with the promoter of the phycocyanin gene (cpc1) that is expressed whatever the incident light conditions. Transcription initiation at the same start point as in vivo was obtained with the Calothrix sp. PCC 7601 purified enzyme and the Escherichia coli core enzyme supplemented with a Calothrix sp. PCC 7601 sigma factor, but not with the E. coli holoenzyme.

Transduction of the light signal during complementary chromatic adaptation in the cyanobacterium Calothrix sp. PCC 7601: DNA-binding proteins and modulation by phosphorylation.

The cyanobacterium Calothrix sp. PCC 7601 can adapt its pigment content in response to changes in the incident light wavelength. It synthesizes, as major light-harvesting pigments, either phycocyanin 2 (PC2, encoded by the cpc2 operon) under red light or phycoerythrin (PE, encoded by the cpeBA operon) under green light conditions. The last step of the signal transduction pathway is characterized by a transcriptional control of the expression of these operons. Partially purified protein extracts were used in gel retardation assays and DNase I footprinting experiments to identify the factors that interact with the promoter region of the cpeBA operon. We found that two proteins, RcaA and RcaB, only detected in extracts of cells grown under green light, behave as positive transcriptional factors for the expression of the cpeBA operon. Treatment of the fractions containing RcaA and RcaB with alkaline phosphatase prevents the binding of RcaA but not of RcaB to the cpeBA promoter region. A post-translational modification of RcaA thus modulates its affinity for DNA.