Applying a circular economy approach, this research explores the use of cheese whey permeate (CWP), by-product of whey ultrafiltration, as cheap substrate for the production of bacterial cellulose (BC) and Sakacin-A, to be used in an antimicrobial packaging material. BC from the acetic acid bacterium Komagataeibacter xylinus was boosted up to 6.77 g/L by supplementing CWP with β-galactosidase. BC was then reduced to nanocrystals (BCNCs, 70% conversion yield), which were then conjugated with Sakacin-A, an anti-Listeria bacteriocin produced by Lactobacillus sakei in a CWP based broth. Active conjugates (75 Activity Units (AU)/mg), an innovative solution for bacteriocin delivery, were then included in a coating mixture applied onto paper sheets at 25 AU/cm2. The obtained antimicrobial food package was found effective in reducing Listeria population in storage trials carried out on a fresh Italian soft cheese (named \"stracchino\") intentionally inoculated with Listeria. Production costs of the active material have been mainly found to be associated (90%) to the purification steps. Setting a maximum prudential 50% cost reduction during process up-scaling, conjugates coating formulation would cost around 0.89 €/A4 sheet. Results represent a practical example of a circular economy production procedure by using a food industry by-product to produce antimicrobials for food preservation.

Lanthipeptides are intriguing peptides known since 1928, the year of penicillin\'s discovery. At that time, they were known as lantibiotics due to their (methyl)lanthionine amino acids and antibacterial activity. Their body of knowledge expanded tremendously over the last few years. Our analysis reveals that Bacteroidetes has a high state of clusters encoding the biosynthesis of class I lanthipeptides. We show that some strains of Pedobacter have a number of LanBs/genome comparable to that of some Actinobacteria. The case study selected was Pedobacter lusitanus NL19. Its clusters identified encode LanBs associated with LanCs as well as orphan LanBs. The first are concomitant with LanT transporters typical of class II lanthipeptides (and not class I), making their clusters into a hybrid class I and class II type. So far, this kind of operon was described only once and is involved in the production of pinensins, the first lanthipeptide with antifungal activity. A particular feature of pinensins is their splitted LanBs and we found that these enzymes are also widely encoded in Bacteroides. The function of a high percentage of proteins predicted to play a role in the production of Pedobacter lanthipeptides is unknown. Other major fraction of these proteins is expected to be enrolled in signal-transduction pathways. We demonstrate that the occurrence of lanthipeptides clusters in the genomes of Gram-negative bacteria is higher than previously reported. More importantly, we show that their genetic background is highly diverse, which is an undeniable foreshadowing of novel peptide structures, biochemistry and biological function.

The gut of the agricultural pest Ceratitis capitata hosts a varied community of bacteria, mainly Enterobacteriaceae, that were implicated in several processes that increase the fitness of the insect. In this study, we investigated the antagonistic activity in vitro of Klebsiella oxytoca strains isolated in the 1990s from the alimentary tract of wild medflies collected from different varieties of fruit trees at diverse localities. Assays were carried out against reference strains (representative of Gram-negative and -positive bacterial species) of the American Type Culture Collection (ATCC). Eight Klebsiella, out of 11, expressed a killing activity against Escherichia coli ATCC 23739, and Enterobacter cloacae ATCC 13047; among the eight strains, at least one showed activity against Salmonella typhimurium ATCC 23853. Genomic DNA derived from all Klebsiella strains was then subjected to PCR amplification using specific primer pairs designed from each of the four bacteriocin (KlebB, C, D, CCL) sequences found so far in Klebsiella. KlebD primer pairs were the only to produce a single product for all strains expressing the killing phenotype in vitro. One of the amplicons was cloned and sequenced; the DNA sequence shows 93% identity with a plasmid-carried colicin-D gene of a strain of Klebsiella michiganensis, and 86% identity with the sequence encoding for the klebicin D activity protein in K. oxytoca. Our work provides the first evidence that dominant symbiotic bacteria associated with wild medfly populations express a killing phenotype that may mediate inter and intraspecies competition among bacterial populations in the insect gut in vivo.

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called \"suicide probes\". The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.

Oral diseases, including dental caries and periodontitis, are among the most prevalent diseases worldwide and develop as a consequence of a microbial dysbiosis. Several bacterial strains are being tested as potential oral health-promoting organisms, but usually they are species isolated from niches other than the site where they must exert its probiotic action, typically from fecal samples. We hypothesize that oral inhabitants associated to health conditions will be more effective than traditional, gut-associated probiotic species in key aspects such as colonization of the oral site where disease takes place or the possession of oral health promoting functions, as well as more practical issues like safety and toxicity, and establishing proper doses for administration. As an example of these active colonizers, we describe the case of Streptococcus dentisani, a new streptococcal species isolated from dental plaque of caries-free individuals. We have detected it in 98% of dental plaque samples from healthy individuals and, as expected, it does not produce any toxic secondary metabolite and does not survive a simulated stomach digestion, preventing potential secondary effects. Besides, this species has a double probiotic action, as it inhibits the growth of major oral pathogens through the production of bacteriocins, and also buffers acidic pH (the primary cause of dental caries) through an arginolytic pathway. We propose the use of S. dentisani as a promising probiotic against tooth decay.

Post-segregational killing (PSK) is a phenotype determined by plasmids using a toxin and an antitoxin gene pair. Loss of the genes depletes the cell\'s reserve of antitoxin and allows the toxin to act upon the cell. PSK benefits mobile elements when it increases reproductive success relative to other mobile competitors. A side effect of PSK is that plasmids become refractory to displacement from the cell during growth as a monoculture. Most PSK systems use a cytoplasmic toxin, but the external toxins of bacteriocins also have a PSK-like effect. It may be that any toxin and antitoxin gene pair can demonstrate PSK when it is on a plasmid. The secreted ribonuclease barnase and its protein inhibitor barstar have features in common with PSK modules, though their native context is chromosomal. We hypothesized that their recruitment to a plasmid could produce an emergent PSK phenotype. Others had shown that secreted barnase could exert a lethal effect on susceptible bacteria similarly to bacteriocins. However, barnase toxicity did not occur under the conditions tested, suggesting that barnase is toxic to neighbouring cells only under very specific conditions. Bacteriocins are only produced under some conditions, and some conditionality on toxin function or release may be advantageous in general to PSKs with external toxins because it would prevent killing of potential plasmid-naive hosts. Too much conditionality, however, would limit how advantageous the gene pair was to mobile elements, making the genes unlikely to be recruited as a PSK system.

Very few studies have been reported on the cytotoxicity and impact of bacteriocins, and especially enterocins, upon eukaryotic cells. In order to gain more information on the safety of bacteriocins, we focused this study on enterocin S37, a bacteriocin produced by Enterococcus faecalis S37. We observed dose-dependent cytotoxicity toward undifferentiated Caco-2/TC7 cells. Moreover, no significant effect on differentiated monolayer Caco-2/TC7 and no apoptotic features were observed when cells were treated with 10 μg/ml of enterocin S37. The results obtained indicate possible safe use of enterocin S37 in the gastrointestinal tract of animals to prevent pathogen invasion and/or infection.

Members of the Gram-positive bacterial genus Streptococcus are a diverse collection of species inhabiting many body sites and range from benign, nonpathogenic species to those causing life-threatening infections. The streptococci are also prolific producers of bacteriocins, which are ribosomally synthesized proteinaceous antibiotics that kill or inhibit species closely related to the producer bacterium. With the emergence of bacterial resistance to conventional antibiotics, there is an impetus to discover, and implement, new and preferably \'natural\' antibiotics to treat or prevent bacterial infections, a niche that bacterial interference therapy mediated by bacteriocins could easily fill. This review focuses on describing the diversity of bacteriocins produced by streptococci and also puts forth a case for Streptococcus salivarius, a nonpathogenic and numerically predominant oral species, as an ideal candidate for development as the model probiotic for the oral cavity. S. salivarius is a safe species that not only produces broad-spectrum bacteriocins but harbors bacteriocin-encoding (and bacteriocin-inducing) transmissible DNA entities (megaplasmids).

Many mutator genes have been characterized in E. coli, but the realization that mutA, the most recent mutator pathway described, encodes for a missense suppressor glycine tRNA caused a real surprise. The connection between expression of mutA and a 10 times increase in the spontaneous mutation rate is not readily explainable. The first attempt to describe the mechanism of action suggested a direct mistranslation of one subunit of polymerase III (PolIII) and the ideal candidate was the epsilon subunit carrying the 3\'-->5\' exonuclease activity. This subunit increases PolIII accuracy about 100 times. However, such direct mistranslation of epsilon was later ruled out when it became clear that all mutA cells express an error-prone form of PolIII. This result could not be reconciled with the very low level of mistranslation (1%) caused by mutA. But there is no need to invoke amino acid misincorporation in epsilon to destroy its activity. On the contrary, I suggest a new way to regulate epsilon amount, based on the reinterpretation of the mutA pathway through the new and puzzling observation that several tRNAs (including mutA which encodes for a glycine missense suppressor tRNA) are complementary to the 5\' end of dnaQ mRNA. Accordingly, I propose that uncharged tRNAs can act as antisense RNAs, decreasing translation of dnaQ and possibly other genes. This could represent a new regulatory function for tRNAs and of course gives a direct and unrecognized link between starvation and mutation rate.

Quorum sensing enables unicellular organisms to behave in a multicellular way by allowing population-wide synchronized adaptive responses that involve modulation of a wide range of physiological responses in a cell density-, cell proximity- or growth phase-dependent manner. Examples of processes modulated by quorum sensing are the development of genetic competence, conjugative plasmid transfer, sporulation and cell differentiation, biofilm formation, virulence response, production of antibiotics, antimicrobial peptides and toxins, and bioluminescence (for reviews see [38]). The cell-to-cell communication strategies involved in these processes are based on the utilization of small signal molecules produced and released into the environment by the microorganisms. These communication molecules are referred to as pheromones and act as chemical messengers that transmit information across space. The extracellular pheromones accumulate in the environment and trigger a response in the target cells when its concentration reaches a certain threshold value. Elucidation of the chemical nature of the pheromones modulating the processes mentioned above reveals that most of them are unmodified peptides, post-translationally modified peptides, N-acyl homoserine lactones, or butyrolactones. Lactone-based pheromones are the preferred communication signals in Gram-negative bacteria (for review see [47,48]), whereas peptide-based pheromones are the predominant extracellular signals among Gram-positive bacteria (for review see [37,61]). However, lactone-based pheromones are utilized as signals that modulate differentiation and secondary metabolism production in Streptomyces (for review see [20]). This review focuses on the major advances and current views of the peptide-pheromone dependent regulatory circuits involved in production of antimicrobial peptides in Gram-positive bacteria.