Alpha-Helical Protein KfrC Acts as a Switch between the Lateral and Vertical Modes of Dissemination of Broad-Host-Range RA3 Plasmid from IncU (IncP-6) Incompatibility Group.

KfrC proteins are encoded by the conjugative broad-host-range plasmids that also encode alpha-helical filament-forming KfrA proteins as exemplified by the RA3 plasmid from the IncU incompatibility group. The RA3 variants impaired in kfrA, kfrC, or both affected the host's growth and demonstrated the altered stability in a species-specific manner. In a search for partners of the alpha-helical KfrC protein, the host's membrane proteins and four RA3-encoded proteins were found, including the filamentous KfrA protein, segrosome protein KorB, and the T4SS proteins, the coupling protein VirD4 and ATPase VirB4. The C-terminal, 112-residue dimerization domain of KfrC was involved in the interactions with KorB, the master player of the active partition, and VirD4, a key component of the conjugative transfer process. In Pseudomonas putida, but not in Escherichia coli, the lack of KfrC decreased the stability but improved the transfer ability. We showed that KfrC and KfrA were involved in the plasmid maintenance and conjugative transfer and that KfrC may play a species-dependent role of a switch between vertical and horizontal modes of RA3 spreading.

Transcriptional Organization of the Stability Module of Broad-Host-Range Plasmid RA3, from the IncU Group.

The broad-host-range (BHR) conjugative plasmids have developed diverse adaptive mechanisms defining the range of their promiscuity. The BHR conjugative RA3 plasmid, the archetype of the IncU group, can transfer between, replicate in, and be maintained in representatives of Alpha-, Beta-, and Gammaproteobacteria Its stability module encompasses ten open reading frames (ORFs) apparently organized into five operons, all transcribed in the same direction from several strong promoters that are tightly regulated either by autorepressors or by global plasmid-encoded regulators. In this paper, we demonstrate that owing to an efficient RNA polymerase (RNAP) read-through, the transcription from the first promoter, orf02p, may continue through the whole module. Moreover, an analysis of mRNA produced from the wild-type (WT) stability module and its deletion variants deprived of particular internal transcription initiation sites reveals that in fact each operon may be transcribed from any upstream promoter, giving rise to multicistronic transcripts of variable length and creating an additional level of gene expression control by transcript dosage adjustment. The gene expression patterns differ among various hosts, indicating that promoter recognition, regulation, and the RNAP read-through mechanisms are modulated in a species-specific manner.IMPORTANCE The efficiently disseminating conjugative or mobilizable BHR plasmids play key roles in the horizontal spread of genetic information between closely related and phylogenetically distant species, which can be harmful from the medical, veterinary, or industrial point of view. Understanding the mechanisms determining the plasmid's ability to function in diverse hosts is essential to help limit the spread of undesirable plasmid-encoded traits, e.g., antibiotic resistance. The range of a plasmid's promiscuity depends on the adaptations of its transfer, replication, and stability functions to the various hosts. IncU plasmids, with the archetype plasmid RA3, are considered to constitute a reservoir of antibiotic resistance genes in aquatic environments; however, the molecular mechanisms determining their adaptability to a broad range of hosts are rather poorly characterized. Here, we present the transcriptional organization of the stability module and show that the gene transcript dosage effect is an important determinant of the stable maintenance of RA3 in different hosts.

The Effect of PUFA-Rich Plant Oils and Bioactive Compounds Supplementation in Pig Diet on Color Parameters and Myoglobin Status in Long-Frozen Pork Meat.

The study evaluated the effect of pig diet supplementation with rapeseed or linseed oil, and vitamin E or selenium, or both vitamin E and selenium on color parameters and myoglobin content of pork Semimembranosus muscle after long-term freezing storage during nine months. The influence of the type of the bioactive compounds added to pig diet on the content of myoglobin or oxymyoglobin, metmyoglobin and deoksymyoglobin in Semimembranosus m. was also assessed. The results indicate that the presence of oils rich in polyunsaturated fatty acids (PUFA) in pig diet improves the color of pork meat. Supplementation of dietary plant oils or dietary oils with antioxidants tended to increase significantly the concentration of oxymyoglobin and decrease the concentration of metmyoglobin in meat compared to the control group. The highest content of oxymyoglobin was observed in meat obtained from pigs fed diets with linseed oil. The best color scores (highest a* parameter) was noted for rapeseed oil group (with no addition of antioxidants). In conclusion, the addition of antioxidants to pigs' forage supplemented with PUFA-rich oils is not recommended in order to improve color of long-term frozen pork.

Antioxidant potential of Haematococcus pluvialis extract rich in astaxanthin on colour and oxidative stability of raw ground pork meat during refrigerated storage.

Astaxanthin is proven to be one of the most potent, naturally occurring antioxidants. A rich source of astaxanthin is algae Haematoccocus pluvialis (H. pluvialis). The aim of the study was to investigate antioxidant effect of H. pluvalis extract added in different levels (0.15, 0.3 or 0.45g/kg of meat) on colour and oxidative stability of raw ground pork meat during refrigerated storage (7days). Obtained data revealed that DPPH scavenging activity of the extract at the concentration of 0.45g/kg of meat was as high as 85%. Moreover, application of higher extract doses (0.3 and 0.45g/kg) delayed lipids oxidation (lower TBARS value than control) and improved colour stability (increased a* colour parameter). Additionally, usage of 0.3 and 0.45g/kg had a positive effect on meat acceptance declared by consumers' at the final day of storage. However, the extract of H. pluvialis had no antimicrobial or antioxidative activity against myoglobin oxidation.

The sensory quality of allergen-controlled, fat-reduced, salt-reduced pork-ostrich sausages during storage.

BACKGROUND: New meat products tailored to consumer health should be characterised by reduced sodium, fat and cholesterol contents and other health-promoting benefits. However, the food sector's greatest challenge is allergen-free production. Consumers are not willing to compromise the sensory quality of meat products for health. The aim of the present study was to analyse the influence of the storage time on the physical properties and consumer acceptance of allergen-controlled, fat-reduced, salt-reduced pork-ostrich sausages. The study focused on pork-ostrich sausages produced in accordance with a new patented technology, which focused on eliminating cross-contamination on-line in the plant, eliminating cross-contamination after preparation, and eliminating spices with high allergy potential. The production was focused on reducing fat (by approximately 50%) and salt (by approximately 30%) levels. RESULTS: No changes in the texture parameters of the sausage were observed during storage time; however, some changes in colour were observed. There were no significant differences in sensory consumer acceptability of pork-ostrich sausage after 14 days of storage; thus, it may be stated that the instrumentally assessed differences in colour did not influence consumer acceptance. CONCLUSION: The applied fat and NaCl reduction in the pork-ostrich sausages contributed to high consumer ratings and was not correlated with saltiness acceptability. © 2017 Society of Chemical Industry.

Mobile antibiotic resistance - the spread of genes determining the resistance of bacteria through food products.

In recent years, more and more antibiotics have become ineffective in the treatment of bacterial nfections. The acquisition of antibiotic resistance by bacteria is associated with circulation of genes in the environment. Determinants of antibiotic resistance may be transferred to pathogenic bacteria. It has been shown that conjugation is one of the key mechanisms responsible for spread of antibiotic resistance genes, which is highly efficient and allows the barrier to restrictions and modifications to be avoided. Some conjugative modules enable the transfer of plasmids even between phylogenetically distant bacterial species. Many scientific reports indicate that food is one of the main reservoirs of these genes. Antibiotic resistance genes have been identified in meat products, milk, fruits and vegetables. The reason for such a wide spread of antibiotic resistance genes is the overuse of antibiotics by breeders of plants and animals, as well as by horizontal gene transfer. It was shown, that resistance determinants located on mobile genetic elements, which are isolated from food products, can easily be transferred to another niche. The antibiotic resistance genes have been in the environment for 30 000 years. Their removal from food products is not possible, but the risks associated with the emergence of multiresistant pathogenic strains are very large. The only option is to control the emergence, selection and spread of these genes. Therefore measures are sought to prevent horizontal transfer of genes. Promising concepts involve the combination of developmental biology, evolution and ecology in the fight against the spread of antibiotic resistance.

Convenient broad-host-range unstable vectors for studying stabilization cassettes in diverse bacteria.

BACKGROUND: Low-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. The efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use. RESULTS: To facilitate such analysis highly unstable, mobilizable, broad-host-range (BHR) vectors based on RK2 replicon were constructed. The vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding ParB homologues capable of spreading on DNA. The xylE or lacZ reporter systems facilitate easy monitoring of plasmid segregation. CONCLUSION: The range of BHR vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species.

Concerted action of NIC relaxase and auxiliary protein MobC in RA3 plasmid conjugation.

Conjugative transfer of the broad-host-range RA3 plasmid, the archetype of the IncU group, relies on the relaxase NIC that belongs to the as yet uncharacterized MOBP4 subfamily. NIC contains the signature motifs of HUH relaxases involved in Tyr nucleophilic attack. However, it differs in the residue involved in His activation for cation coordination and was shown here to have altered divalent cation requirements. NIC is encoded in the mobC-nic operon preceded directly by oriT, where mobC encodes an auxiliary transfer protein with a dual function: autorepressor and stimulator of conjugative transfer. Here an interplay between MobC and NIC was demonstrated. MobC is required for efficient NIC cleavage of oriT in supercoiled DNA whereas NIC assists MobC in repression of the mobC-nic operon. A 7-bp arm of IR3 (IR3a) was identified as the binding site for NIC and the crucial nucleotides in IR3a for NIC recognition were defined. Fully active oriTRA3 was delineated to a 47-bp DNA segment encompassing a conserved cleavage site sequence, the NIC binding site IR3a and the MobC binding site OM . This highly efficient RA3 conjugative system with defined requirements for minimal oriT could find ample applications in biotechnology and computational biology where simple conjugative systems are needed.

Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family).

The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nicoperon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions in Escherichia coli, since its overexpression in transnegatively interferes with mini-RA3 replication and stable maintenance of RA3.

MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer.

BACKGROUND: The IncU conjugative transfer module represents highly efficient promiscuous system widespread among conjugative plasmids of different incompatibility groups. Despite its frequent occurrence the mechanisms of relaxosome formation/action are far from understood. Here we analyzed the putative transfer auxiliary protein MobC of the conjugative plasmid RA3 from the IncU incompatibility group. RESULTS: MobC is a protein of 176 amino acids encoded in the bicistronic operon mobC-nic adjacent to oriT. MobC is homologous to prokaryotic transcription factors of the ribbon-helix-helix (RHH) superfamily. Conserved LxxugxNlNQiaxxLn motif clusters MobC with the clade of conjugative transfer auxilliary proteins of MobP relaxases. MobC forms dimers in solution and autoregulates the expression of mobCp by binding to an imperfect palindromic sequence (OM) located between putative -35 and -10 motifs of the promoter. Medium-copy number test plasmid containing the oriT-mobCp region is mobilized with a high frequency by the RA3 conjugative system. The mutations introduced into OM that abolished MobC binding in vitro decreased 2-3 fold the frequency of mobilization of the test plasmids. The deletion of OM within the RA3 conjugative module had no effect on transfer if the mobC-nic operon was expressed from the heterologous promoter. If only nic was expressed from the heterologous promoter (no mobC) the conjugative transfer frequency of such plasmid was 1000-fold lower. CONCLUSION: The MobC is an auxiliary transfer protein of dual function. It autoregulates the expression of mobC-nic operon while its presence significantly stimulates transfer efficiency.