This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
Listeria monocytogenes is a food-borne pathogen which is the causative agent of listeriosis and can be divided into three evolutionary lineages I, II and III. While all strains possess the well established virulence factors associated with the Listeria pathogenicity island I (LIPI-1), lineage I strains also possess an additional pathogenicity island designated LIPI-3 which encodes listeriolysin S (LLS), a post-translationally modified cytolytic peptide. Up until now, this pathogenicity island has been identified exclusively in a subset of lineage I isolates of the pathogen Listeria monocytogenes.
In total 64 L. innocua strains were screened for the presence of LIPI-3. Here we report the identification of an intact LIPI-3 in 11 isolates of L. innocua and the remnants of the cluster in several others. Significantly, we can reveal that placing the L. innocua lls genes under the control of a constitutive promoter results in a haemolytic phenotype, confirming that the cluster is capable of encoding a functional haemolysin.
Although the presence of the LIPI-3 gene cluster is confined to lineage I isolates of L. monocytogenes, a corresponding gene cluster or its remnants have been identified in many L. innocua strains.
Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183–207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.
Streptolysin S (SLS) is a potent cytolytic toxin and virulence factor produced by nearly all Streptococcus pyogenes strains. Despite a 100-year history of research on this toxin, it has only recently been established that SLS represents the archetypal example of an extended family of post-translationally modified virulence factors also produced by some other streptococci and Gram-positive pathogens, such as Listeria monocytogenes and Clostridium botulinum. In this Review we describe the identification, genetics, biochemistry and various functions of SLS. We also discuss the shared features of the virulence-associated SLS-like peptides, as well as their place within the rapidly expanding family of thiazole/oxazole-modified microcins (TOMMs).
The renin angiotensin system (RAS) plays an important role in wound repair; however, little is known pertaining to RAS expression in response to thermal and the combination of radiation plus burn injury (CRBI). The purpose of this study was to test the hypothesis that thermal injury modifies expression of RAS components and CRBI delayed this up-regulation of RAS. Skin from uninjured mice was compared to mice receiving local thermal injury or CRBI (injury site). Skin was analyzed for gene and protein expression of RAS components. There was an initial increase in the expression of various components of RAS following thermal injury. However, in the higher CRBI group there is an initial decrease in AT1b (vasoconstriction, pro-proliferative) AT2 (vasodilation, differentiation) and Mas (vasodilation, anti-inflammatory) gene expression. This corresponded with a delay and decrease in AT1, AT2 and MAS protein expression in fibroblasts and keratinocytes. The reduction in RAS receptor positive fibroblasts and keratinocytes correlated with a reduction in collagen deposition and keratinocyte infiltration into the wounded area resulting in a delay of re-epithelialization following CRBI. These data support the hypothesis that delayed wound healing observed in subjects following radiation exposure may be in part due to decreased expression of RAS.
angiotensin; combined radiation and burn injury; wound healing
The human intestinal microbiota is one of the most densely populated ecosystems on Earth, containing up to 1013 bacteria/g and in some respects can be considered an organ itself given its role in human health. Bacteriophages (phages) are the most abundant replicating entities on the planet and thrive wherever their bacterial hosts exist. They undoubtedly influence the dominant microbial populations in many ecosystems including the human intestine. Within this setting, lysogeny appears to be the preferred life cycle, presumably due to nutrient limitations and lack of suitable hosts protected in biofilms, hence the predator/prey dynamic observed in many ecosystems is absent. On the other hand, free virulent phages in the gut are more common among sufferers of intestinal diseases and have been shown to increase with antibiotic usage. Many of these phages evolve from prophages of intestinal bacteria and emerge under conditions where their bacterial hosts encounter stress suggesting that prophages can significantly alter the microbial community composition. Based on these observations, we propose the “community shuffling” model which hypothesizes that prophage induction contributes to intestinal dysbiosis by altering the ratio of symbionts to pathobionts, enabling pathobiont niche reoccupation. The consequences of the increased phage load on the mammalian immune system are also addressed. While this is an area of intestinal biology which has received little attention, this review assembles evidence from the literature which supports the role of phages as one of the biological drivers behind the composition of the gut microbiota.
gut; microbiota; bacteriophages; phages; prophages; induction; community shuffling
Obesity is associated with a number of serious health consequences, including type 2 diabetes, cardiovascular disease and a variety of cancers among others and has been repeatedly shown to be associated with a higher risk of mortality. The relatively recent discovery that the composition and metabolic activity of the gut microbiota may affect the risk of developing obesity and related disorders has led to an explosion of interest in this distinct research field. A corollary of these findings would suggest that modulation of gut microbial populations can have beneficial effects with respect to controlling obesity. In this addendum, we summarize our recent data, showing that therapeutic manipulation of the microbiota using different antimicrobial strategies may be a useful approach for the management of obesity and metabolic conditions. In addition, we will explore some of the mechanisms that may contribute to microbiota-induced susceptibility to obesity and metabolic diseases.
obesity; antimicrobials; gut microbiota; firmicutes; metabolic disease
Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo, we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P16S, cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R2 > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (109 CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ∼400- and ∼900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living animals in real time.
The foodborne pathogen Listeria monocytogenes has the capacity to survive and grow in a diverse range of natural environments. The transition from a food environment to the gastrointestinal tract begins a process of adaptation that may culminate in invasive systemic disease. Here we describe recent advances in our understanding of how L. monocytogenes adapts to the gastrointestinal environment prior to initiating systemic infection. We will discuss mechanisms used by the pathogen to survive encounters with acidic environments (which include the glutamate decarboxylase and arginine deiminase systems), and those which enable the organism to cope with bile acids (including bile salt hydrolase) and competition with the resident microbiota. An increased understanding of how the pathogen survives in this environment is likely to inform the future design of novel prophylactic approaches that exploit specific pharmabiotics; including probiotics, prebiotics, or phages.
Listeria; stress; acid; bile; gastrointestinal; virulence; pathogenesis; infection
Functional environmental screening of metagenomic libraries is a powerful means to identify and assign function to novel genes and their encoded proteins without any prior sequence knowledge. In the current study we describe the identification and subsequent analysis of a salt-tolerant clone from a human gut metagenomic library. Following transposon mutagenesis we identified an unknown gene (stlA, for “salt tolerance locus A”) with no current known homologues in the databases. Subsequent cloning and expression in Escherichia coli MKH13 revealed that stlA confers a salt tolerance phenotype in its surrogate host. Furthermore, a detailed in silico analysis was also conducted to gain additional information on the properties of the encoded StlA protein. The stlA gene is rare when searched against human metagenome datasets such as MetaHit and the Human Microbiome Project and represents a novel and unique salt tolerance determinant which appears to be found exclusively in the human gut environment.
Due to the ongoing problem of recurrence of Clostridium difficile-associated diarrhea following antibiotic treatment, there is an urgent need for alternative treatment options. We assessed the MICs of five antimicrobials singly and in combinations against a range of C. difficile clinical isolates. Ramoplanin-actagardine combinations were particularly effective, with partial synergistic/additive effects observed against 61.5% of C. difficile strains tested.
Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.
Bacteriocin production is an important probiotic trait of intestinal bacteria. In this study, we identify a new type of bacteriocin, bactofencin A, produced by a porcine intestinal isolate Lactobacillus salivarius DPC6502, and assess its potency against pathogenic species including Staphylococcus aureus and Listeria monocytogenes. Genome sequencing of the bacteriocin producer revealed bfnA, which encodes the mature and highly basic (pI 10.59), 22-amino-acid defensin-like peptide. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectral analysis determined that bactofencin A has a molecular mass of 2,782 Da and contains two cysteine residues that form an intramolecular disulfide bond. Although an ABC transporter and transport accessory protein were also present within the bacteriocin gene cluster, a classical bacteriocin immunity gene was not detected. Interestingly, a dltB homologue was identified downstream of bfnA. DltB is usually encoded within the dlt operon of many Gram-positive bacteria. It is responsible for d-alanylation of teichoic acids in the cell wall and has previously been associated with bacterial resistance to cationic antimicrobial peptides. Heterologous expression of this gene conferred bactofencin A-specific immunity on sensitive strains of L. salivarius and S. aureus (although not L. monocytogenes), establishing its role in bacteriocin immunity. An analysis of the distribution of bfnA revealed that it was present in four additional isolates derived from porcine origin and absent from five human isolates, suggesting that its distribution is host specific. Given its novelty, we anticipate that bactofencin A represents the prototype of a new class of bacteriocins characterized as being cationic, with a DltB homologue providing a cognate immunity function.
This study describes the identification, purification, and characterization of bactofencin A, a novel type of bacteriocin produced by L. salivarius DPC6502. Interestingly, bactofencin A is not similar to any other known bacteriocin but instead shares similarity with eukaryotic cationic antimicrobial peptides, and here, we demonstrate that it inhibits two medically significant pathogens. Genome sequence analysis of the producing strain also revealed the presence of an atypical dltB homologue in the bacteriocin gene cluster, which was lacking a classical bacteriocin immunity gene. Furthermore, cloning this gene rendered sensitive strains resistant to the bacteriocin, thereby establishing its role in providing cognate bacteriocin immunity. Four additional L. salivarius isolates, also of porcine origin, were found to contain the bacteriocin biosynthesis genes and successfully produced bactofencin A, while these genes were absent from five human-derived strains investigated.
There is an increasing need for alternatives to antibiotics for promoting animal health, given the increasing problems associated with antibiotic resistance. In this regard, we evaluated spent cider yeast as a potential probiotic for modifying the gut microbiota in weanling pigs using pyrosequencing of 16S rRNA gene libraries.
Methodology and Principal Findings
Piglets aged 24–26 days were assigned to one of two study groups; control (n = 12) and treatment (n = 12). The control animals were fed with a basal diet and the treatment animals were fed with basal diet in combination with cider yeast supplement (500 ml cider yeast containing ∼7.6 log CFU/ml) for 21 days. Faecal samples were collected for 16s rRNA gene compositional analysis. 16S rRNA compositional sequencing analysis of the faecal samples collected from day 0 and day 21 revealed marked differences in microbial diversity at both the phylum and genus levels between the control and treatment groups. This analysis confirmed that levels of Salmonella and Escherichia were significantly decreased in the treatment group, compared with the control (P<0.001). This data suggest a positive influence of dietary supplementation with live cider yeast on the microbial diversity of the pig distal gut.
The effect of dietary cider yeast on porcine gut microbial communities was characterized for the first time using 16S rRNA gene compositional sequencing. Dietary cider yeast can potentially alter the gut microbiota, however such changes depend on their endogenous microbiota that causes a divergence in relative response to that given diet.
Metagenomics is a powerful tool that allows for the culture-independent analysis of
complex microbial communities. One of the most complex and dense microbial ecosystems
known is that of the human distal colon, with cell densities reaching up to
1012 per gram of faeces. With the majority of species as yet uncultured,
there are an enormous number of novel genes awaiting discovery. In the current study, we
conducted a functional screen of a metagenomic library of the human gut microbiota for
potential salt-tolerant clones. Using transposon mutagenesis, three genes were identified
from a single clone exhibiting high levels of identity to a species from the genus
Collinsella (closest relative being Collinsella aerofaciens)
(COLAER_01955, COLAER_01957 and COLAER_01981), a high G+C, Gram-positive member of
the Actinobacteria commonly found in the human gut. The encoded proteins exhibit a strong
similarity to GalE, MurB and MazG. Furthermore, pyrosequencing and bioinformatic analysis
of two additional fosmid clones revealed the presence of an additional galE and
mazG gene, with the highest level of genetic identity to Akkermansia
muciniphila and Eggerthella sp. YY7918, respectively. Cloning and
heterologous expression of the genes in the osmosensitive strain, Escherichia
coli MKH13, resulted in increased salt tolerance of the transformed cells. It is
hoped that the identification of atypical salt tolerance genes will help to further
elucidate novel salt tolerance mechanisms, and will assist our increased understanding how
resident bacteria cope with the osmolarity of the gastrointestinal tract.
; human gut microbiome; metagenomics; salt tolerance
The emergence of bacterial drug resistance encourages the re-evaluation of the potential of existing antimicrobials. Lantibiotics are post-translationally modified, ribosomally synthesised antimicrobial peptides with a broad spectrum antimicrobial activity. Here, we focussed on expanding the potential of lacticin 3147, one of the most studied lantibiotics and one which possesses potent activity against a wide range of Gram positive species including many nosocomial pathogens. More specifically, our aim was to investigate if lacticin 3147 activity could be enhanced when combined with a range of different clinical antibiotics.
Initial screening revealed that polymyxin B and polymyxin E (colistin) exhibited synergistic activity with lacticin 3147. Checkerboard assays were performed against a number of strains, including both Gram positive and Gram negative species. The resultant fractional inhibitory concentration (FIC) index values established that, while partial synergy was detected against Gram positive targets, synergy was obvious against Gram negative species, including Cronobacter and E. coli.
Combining lacticin 3147 with low levels of a polymyxin could provide a means of broadening target specificity of the lantibiotic, while also reducing polymyxin use due to the lower concentrations required as a result of synergy.
Antimicrobial; Synergy; Lantibiotic; Bacteriocin; Lacticin 3147; Polymyxin
A recent comparative genomic hybridization study in our laboratory revealed considerable plasticity within the bacteriocin locus of gastrointestinal strains of Lactobacillus salivarius. Most notably, these analyses led to the identification of two novel unmodified bacteriocins, salivaricin L and salivaricin T, produced by the neonatal isolate L. salivarius DPC6488 with immunity, regulatory and export systems analogous to those of abp118, a two-component bacteriocin produced by the well characterized reference strain L. salivarius UCC118. In this addendum we discuss the intraspecific diversity of our seven bacteriocin-producing L. salivarius isolates on a genome-wide level, and more specifically, with respect to their salivaricin loci.
Lactobacillus salivarius; bacteriocin; comparative genomic hybridization; probiotic; salivaricin
We demonstrate that growth of Cronobacter sakazakii in the presence of acetate as a carbon source promotes loss of RpoS, with a consequent reduction in stress tolerance. This suggests that C. sakazakii is capable of regulating cell fitness through mutation of the rpoS gene.
The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post-translationally modified antimicrobial peptides, Ltnα and Ltnβ, which act synergistically against a wide range of Gram-positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering.
Funding Information Work in the authors' laboratory is supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H and R.P.R. with SFI Principal Investigator funding.
Kefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists.
The increasing interest in the human microbiota raises some interesting questions about the terminology we use to describe some of the structures and strategies employed by commensal and pathogenic microbes to compete in these complex biological ecosystems. For example, all microbes arriving in the alimentary tract face the task of surviving passage through the stomach, coping with bile, interacting with the immune system, competing with the established microbiota, and obtaining sufficient nutrients to gain a foothold in this hostile environment. It is not surprising then that many gastrointestinal microbes (both pathogens and commensals) use similar strategies to overcome the challenges associated with this particular biological niche. Given that many of these structures and strategies were discovered and characterized in pathogens and because they often play important roles in establishing and maintaining an infection, they have often been characterized as virulence factors. It would be misleading to describe the same strategies and structures found in harmless commensals as “virulence factors,” since they represent a sine qua non for life in the gastrointestinal tract. It may be time to reconsider and refer to them as “niche factors,” both in terms of providing scientific accuracy but also in light of the growing interest in using gut microbes as probiotics, where the distinction between virulence factors and niche factors is likely to be very important from a regulatory perspective.
Mesenchymal stem cells (MSCs) are capable of regenerative and immunomodulatory functions in cell-based therapies in a variety of human diseases and injuries; however, their therapeutic efficacy and potential side effects remain major obstacles in clinical applications. We report here a 3D spheroid culture approach to optimize stem cell properties and therapeutic effects of human gingiva-derived mesenchymal stem cells (GMSCs) in mitigation of experimental oral mucositis. Under growth condition of ultra-low attachment, GMSCs spontaneously aggregated into 3D spheroids and exhibited distinct early stem cell phenotype characterized by elevated expression Stro-1 and CXC chemokine receptor 4 (CXCR-4) as well as OCT-4 and Nanog, 2 important transcriptional factors relevant to stem cell properties, and decreased expression of MSC-associated markers, including CD29, CD90, and CD105. Functionally, spheroid GMSCs are capable of enhanced multipotency and augmented secretion of several chemokines and cytokines relevant to cell migration, survival, and angiogenesis. More importantly, spheroid GMSCs expressed increased levels of reactive oxygen species, hypoxia-inducible factor (HIF)-1 and -2α, and manganese superoxide dismutase, which correlated with improved resistance to oxidative stress-induced apoptosis. Using an in vivo murine model of chemotherapy-induced oral mucositis, we demonstrated that spheroid-derived GMSCs possessed better therapeutic efficacy than their adherent cells in reversing body weight loss and promoting the regeneration of disrupted epithelial lining of the mucositic tongues. These findings suggest that 3D spheroid culture allows early stemness preservation and potentially precondition GMSCs for enhanced mitigation of oral mucositis.
The lantibiotic lacticin 3147 has been the focus of much research due to its broad spectrum of activity against many microbial targets, including drug-resistant pathogens. In order to protect itself, a lacticin 3147 producer must possess a cognate immunity mechanism. Lacticin 3147 immunity is provided by an ABC transporter, LtnFE, and a dedicated immunity protein, LtnI, both of which are capable of independently providing a degree of protection. In the study described here, we carried out an in-depth investigation of LtnI structure-function relationships through the creation of a series of fusion proteins and LtnI determinants that have been the subject of random and site-directed mutagenesis. We establish that LtnI is a transmembrane protein that contains a number of individual residues and regions, such as those between amino acids 20 and 27 and amino acids 76 and 83, which are essential for LtnI function. Finally, as a consequence of the screening of a bank of 28,000 strains producing different LtnI derivatives, we identified one variant (LtnI I81V) that provides enhanced protection. To our knowledge, this is the first report of a lantibiotic immunity protein with enhanced functionality.
It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.
Betaine uptake in Listeria monocytogenes is mediated by three independent transport systems, the simplest of which in genetic terms is the secondary transporter BetL. Using a random mutagenesis approach, based on the E. coli XL1 Red mutator strain, we identified a single point mutation in a putative promoter region upstream of the BetL coding region which leads to a significant increase in betL transcript levels under osmo- and chill-stress conditions and a concomitant increase in stress tolerance. Furthermore, the mutation appears to counter the heretofore unreported “twisted” cell morphology observed for L. monocytogenes grown at elevated osmolarities in tryptone soy broth.
Listeria; osmotolerance; chill-tolerance; salt stress; twisted cells