An oxidation resistant pediocin PA-1 derivative and penocin A display effective anti-Listeria activity in a model human gut environment.

Pediocin PA-1 is a class IIa bacteriocin that is particularly effective against the foodborne pathogen Listeria monocytogenes. The loss of activity of PA-1 pediocin due to methionine oxidation is one of the challenges that limit the wider application of the bacteriocin. In this study, we heterologously expressed an oxidation resistant form of pediocin PA-1, i.e., pediocin M31L, and compared its activity to that of native pediocin PA-1 and to penocin A, a pediocin-like bacteriocin that displays a narrower antimicrobial spectrum. Minimal inhibitory concentration assays revealed that pediocin M31L was as effective as PA-1 and more effective than synthetic penocin A against Listeria with negligible activity against a range of obligate anaerobic commensal gut bacterial species. The anti-Listeria activity of these pediocins was also assessed in a simulated human distal colon model assay using the L. monocytogenes, spiked at 6.5 ± 0.13 Log CFU/mL, as a bioindicator. At 24 h, pediocin M31L and penocin A (2.6 µM) reduced Listeria counts to 3.5 ± 0.4 and 3.64 ± 0.62 Log CFU/mL, respectively, whereas Listeria counts were considerably higher, i.e. 7.75 ± 0.43 Log CFU/mL, in the non-bacteriocin-containing control. Ultimately, it was established that synthetic penocin A and the stable pediocin M31L derivative, heterologously produced, display effective anti-Listeria activity in a human gut environment.

Antimicrobials for food and feed; a bacteriocin perspective.

Bacteriocins are natural antimicrobials that have been consumed via fermented foods for millennia and have been the focus of renewed efforts to identify novel bacteriocins, and their producing microorganisms, for use as food biopreservatives and other applications. Bioengineering bacteriocins or combining bacteriocins with multiple modes of action (hurdle approach) can enhance their preservative effect and reduces the incidence of antimicrobial resistance. In addition to their role as food biopreservatives, bacteriocins are gaining credibility as health modulators, due to their ability to regulate the gut microbiota, which is strongly associated with human wellbeing. Indeed the strengthening link between the gut microbiota and obesity make bacteriocins ideal alternatives to Animal Growth Promoters (AGP) in animal feed also. Here we review recent advances in bacteriocin research that will contribute to the development of functional foods and feeds as a consequence of roles in food biopreservation and human/animal health.

Heterologous expression of proteorhodopsin enhances H2 production in Escherichia coli when endogenous Hyd-4 is overexpressed.

Proteorhodopsin (PR) is a light harvesting protein widely distributed among bacterioplankton that plays an integral energetic role in a new pathway of marine light capture. The conversion of light into chemical energy in non-chlorophyll-based bacterial systems could contribute to overcoming thermodynamic and metabolic constraints in biofuels production. In an attempt to improve biohydrogen production yields, H2 evolution catalyzed by endogenous hydrogenases, Hyd-3 and/or Hyd-4, was measured when recombinant proteorhodopsin (PR) was concomitantly expressed in Escherichia coli cells. Higher amounts of H2 were obtained with recombinant cells in a light and chromophore dependent manner. This effect was only observed when HyfR, the specific transcriptional activator of the hyf operon encoding Hyd-4 was overexpressed in E. coli, suggesting that an excess of protons generated by PR activity could increase hydrogen production by Hyd-4 but not by Hyd-3. Although many of the subunits of Hyd-3 and Hyd-4 are very similar, Hyd-4 possesses three additional proton-translocating NADH-ubiquinone oxidoreductase subunits, suggesting that it is dependent upon ΔµH(+). Altogether, these results suggest that protons generated by proteorhodopsin in the periplasm can only enhance hydrogen production by hydrogenases with associated proton translocating subunits.

Phosphate deficiency (N/P 40:1) induces mcyD transcription and microcystin synthesis in Microcystis aeruginosa PCC7806.

A real-time RT-PCR analysis of the transcriptional response to phosphate availability of the mcyD gene and microcystin-LR synthesis in Microcystis aeruginosa PCC7806 revealed that no significant changes were observed in the relative quantification of mcyD under excess phosphate (N/P = 1:1), whereas in deficiency of this nutrient (N/P = 40:1), a steady increase of mcyD during the exponential growth phase was detected, showing a maximal level on the 7th day of growth with a 6.8-fold increase over the control cells. The microcystin content in phosphate deficient cells correlates with the trend of mcyD transcription observed. Also, in this work we demonstrate that under phosphate deficiency conditions with a ratio of 40:1 N/P, the growth of M. aeruginosa PCC7806 was not affected when compared to control and phosphate excess samples. When blooms occur, the nutrients become exhausted and therefore phosphate availability will be scarce. In such a complex scenario, microcystin synthesis could be a response to phosphate deficiency, among other stress parameters.

2-oxoglutarate enhances NtcA binding activity to promoter regions of the microcystin synthesis gene cluster.

The binding affinity of NtcA towards promoter regions of the microcystin gene cluster from Microcystis aeruginosa PCC 7806 has been analyzed by band-shift assay (EMSA). The key nitrogen transcriptional regulator exhibits affinity for two fragments of the bidirectional mcyDA promoter, as well as for promoter regions of mcyE and mcyH. The presence of 2-oxoglutarate increased by 2.5 fold the affinity of NtcA for the mcyA promoter region. The 2-oxoglutarate effect peaked at 0.8 mM, a physiological concentration for this compound under nitrogen-limiting conditions. The results suggest that the 2-oxoglutarate level, as a signal of the C to N balance of the cells, regulates the microcystin gene cluster.

Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRP30C.

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.

A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57.

Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6xHis-Ki-1/57(122-413) and 6xHis-Ki-1/57(264-413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein beta subunit as template. Our model suggests the family-typical seven-bladed beta-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.

Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins.

Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.