When a plasmid containing restriction–modification (R–M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R–M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R–M systems, specialized Control (C) proteins coordinate expression of the R and the M genes. C proteins bind to DNA sequences called C-boxes and activate expression of their cognate R genes and inhibit the M gene expression, however the mechanisms remain undefined. Here, we studied the regulation of gene expression in the C protein-dependent EcoRV system. We map the divergent EcoRV M and R gene promoters and we define the site of C protein-binding that is sufficient for activation of the EcoRV R transcription.

Four xylanases of Cellulomonas flavigena were cloned, expressed in Escherichia coli and purified. Three enzymes (CFXyl1, CFXyl2, and CFXyl4) were from the GH10 family, while CFXyl3 was from the GH11 family. The enzymes possessed moderate temperature stability and a neutral pH optimum. The enzymes were more stable at alkaline pH values. CFXyl1 and CFXyl2 hydrolyzed xylan to form xylobiose, xylotriose, xylohexaose, xylopentaose, and xylose, which is typical for GH10. CFXyl3 (GH11) and CFXyl4 (GH10) formed the same xylooligosaccharides, but xylose was formed in small amounts. The xylanases made efficient saccharification of rye, wheat and oat, common components of animal feed, which indicates their high biotechnological potential.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0–7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed K_m 2.16 mg/mL and V_max 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

ФГБНУ "ВНИИМП им. В.М. Горбатова"

The residual content of the only carbon source increases at the end of the exponential growth phase when P. putida BS3701 cells are cultured under nitrogen deficiency. Moreover, the mRNA quantity of some genes involved in carbon catabolism decreases. There are no available data on regulators capable of stimulating the target mRNA degradation in pseudomonads under nitrogen deficiency in the literature. We have applied both comparative genomics methods and a number of biomolecular ones to identify new small P. putida BS3701 RNAs. This is due to the fact that the formation of ncRNA-mRNA duplexes, recognized by RNases, is one of the main mechanisms for reducing the number of transcripts in a cell. We have used the following selection criteria: 1) the presence of the interest gene of the NtrC binding site and a sigma54-dependent promoter in the upstream area, 2) the conservative location of the interest gene in the P. putida and P. aeruginosa genomes, 3) increase in the product quantity of the interest gene in a cell under nitrogen deficiency on two different carbon sources (succinate and naphthalene), 4) the product detection of the interest gene in a fraction enriched with small RNAs. The expression of two new small RNAs has increased twice or more times under nitrogen deficiency on both succinate and naphthalene: expression of ng171 and ng379. ng379 contains narK RNA motif.