The four-plasmid complement of the raw milk cheese isolate Lactococcus lactis subsp. lactis biovar diacetylactis DPC3901 was sequenced, and some genetic features were functionally analyzed. The complete sequences of pVF18 (18,977 bp), pVF21 (21,739 bp), pVF22 (22,166 bp), and pVF50 (53,876 bp) were obtained. Each plasmid contained genes not previously described for Lactococcus, in addition to genes associated with plant-derived lactococcal strains. Most of the novel genes were found on pVF18 and encoded functions typical of bacteria associated with plants, such as activities of plant cell wall modification (orf11 and orf25). In addition, a predicted high-affinity regulated system for the uptake of cobalt was identified (orf19 to orf21 [orf19-21]), which has a single database homolog on a plant-derived Leuconostoc plasmid and whose functionality was demonstrated following curing of pVF18. pVF21 and pVF22 encode additional metal transporters, which, along with orf19-21 of pVF18, could enhance host ability to uptake growth-limiting amounts of biologically essential ions within the soil. In addition, vast regions from pVF50 and pVF21 share significant homology with the plant-derived lactococcal plasmid pGdh442, which is indicative of extensive horizontal gene transfer and recombination between these plasmids and suggests a common plant niche for their hosts. Phenotypes associated with these regions include glutamate dehydrogenase activity and Na(+) and K(+) transport. The presence of numerous plant-associated markers in L. lactis DPC3901 suggests a plant origin for the raw milk cheese isolate and provides for the first time the genetic basis to support the concept of the plant-milk transition for Lactococcus strains.
The high mortality rate associated with Listeria monocytogenes and its ability to adapt to the harsh conditions employed in food processing has ensured that this pathogen remains a serious problem in the ready-to-eat food sector. Bacteriophage-derived enzymes can be applied as biocontrol agents to target specific foodborne pathogens. We investigated the ability of a listeriophage endolysin and derivatives thereof, fused to polyhydroxyalkanoate bionanoparticles (PHA_BNPs), to lyse and inhibit the growth of L. monocytogenes. Turbidity reduction assays confirmed the lysis of L. monocytogenes cells at 37 °C upon addition of the tailored BNPs. The application of BNPs also resulted in the growth inhibition of L. monocytogenes. BNPs displaying only the amidase domain of the phage endolysin were more effective at inhibiting growth under laboratory conditions (37 °C, 3 × 107 CFU/mL) than BNPs displaying the full-length endolysin (89% vs. 83% inhibition). Under conditions that better represent those found in food processing environments (22 °C, 1 × 103 CFU/mL), BNPs displaying the full-length endolysin demonstrated a greater inhibitory effect compared to BNPs displaying only the amidase domain (61% vs. 54% inhibition). Our results demonstrate proof-of-concept that tailored BNPs displaying recombinant listeriophage enzymes are active inhibitors of L. monocytogenes.
The aim of this study was to use comparative modeling to predict the three-dimensional structure of the CHAP(K) protein (cysteine, histidine-dependent amidohydrolase/peptidase domain of the LysK endolysin, derived from bacteriophage K). Iterative PSI-BLAST searches against the Protein Data Bank (PDB) and nonredundant (nr) databases were used to populate a multiple alignment for analysis using the T-Coffee Expresso server. A consensus Maximum Parsimony phylogenetic tree with a bootstrap analysis setting of 1,000 replicates was constructed using MEGA4. Structural templates relevant to our target (CHAP(K)) were identified, processed in Expresso and used to generate a 3D model in the alignment mode of SWISS-MODEL. These templates were also processed in the I-TASSER web server. A Staphylococcus saprophyticus CHAP domain protein, 2K3A, was identified as the structural template in both servers. The I-TASSER server generated the CHAP(K) model with the best bond geometries when analyzed using PROCHECK and the most logical organization of the structure. The predicted 3D model indicates that CHAP(K) has a papain-like fold. Circular dichroism spectropolarimetry also indicated that CHAP(K) has an αβ fold, which is consistent with the model presented. The putative active site maintained a highly conserved Cys54-His117-Glu134 charge relay and an oxyanion hole residue Asn136. The residue triplet, Cys-His-Glu, is known to be a viable proteolytic triad in which we predict the Cys residue is used in a nucleophilic attack on peptide bonds at a specific site in the pentaglycine cross bridge of staphylococcal cell wall peptidoglycan. Use of comparative modeling has allowed approximation of the 3D structure of CHAP(K) giving information on the structure and an insight into the binding and active site of the catalytic domain. This may facilitate its development as an alternative antibacterial agent.
Lactococcus lactis is predominantly associated with dairy fermentations, but evidence suggests that the domesticated organism originated from a plant niche. L. lactis possesses an unusual taxonomic structure whereby strain phenotypes and genotypes often do not correlate, which in turn has led to confusion in L. lactis classification. A bank of L. lactis strains was isolated from various nondairy niches (grass, vegetables, and bovine rumen) and was further characterized on the basis of key technological traits, including growth in milk and key enzyme activities. Phenotypic analysis revealed all strains from nondairy sources to possess an L. lactis subsp. lactis phenotype (lactis phenotype); however, seven of these strains possessed an L. lactis subsp. cremoris genotype (cremoris genotype), determined by two separate PCR assays. Multilocus sequence typing (MLST) showed that strains with lactis and cremoris genotypes clustered together regardless of habitat, but it highlighted the increased diversity that exists among "wild" strains. Calculation of average nucleotide identity (ANI) and tetranucleotide frequency correlation coefficients (TETRA), using the JSpecies software tool, revealed that L. lactis subsp. cremoris and L. lactis subsp. lactis differ in ANI values by ∼14%, below the threshold set for species circumscription. Further analysis of strain TIFN3 and strains from nonindustrial backgrounds revealed TETRA values of <0.99 in addition to ANI values of <95%, implicating that these two groups are separate species. These findings suggest the requirement for a revision of L. lactis taxonomy.
The lantibiotics are a group of ribosomally synthesised, post-translationally modified peptides containing unusual amino acids, such as dehydrated and lanthionine residues. This group of bacteriocins has attracted much attention in recent years due to the success of the well characterised lantibiotic, nisin, as a food preservative. Numerous other lantibiotics have since been identified and can be divided into two groups on the basis of their structures, designated type-A and type-B. To date, many of these lantibiotics have undergone extensive characterisation resulting in an advanced understanding of them at both the structural and mechanistic level. This review outlines some of the more recent developments in the biochemistry, genetics and mechanism of action of these peptides.
Mannitol is a sugar alcohol, or polyol, widely used in the food industry because of its low-calorie properties. Industrial production of mannitol is difficult and expensive. However, certain bacterial species are known to produce mannitol naturally, including certain lactic acid bacteria and fructophilic lactic acid bacteria (LAB). In this study, bacterial strains isolated from fructose-rich sources, including flowers, leaves, and honey, were identified by 16S rRNA sequence analysis as Leuconostoc, Fructobacillus, Lactococcus, and Lactobacillus species and 4 non-LAB species. DNA profiles generated by pulsed-field gel electrophoresis discriminated 32 strains of Leuconostoc mesenteroides and 6 Fructobacillus strains. Out of 41 LAB strains isolated, 32 were shown to harbor the mdh gene, which encodes the mannitol dehydrogenase enzyme, and several showed remarkable fructose tolerance even at 50% fructose concentrations, indicating their fructophilic nature. Several of the strains isolated, including Leuconostoc mesenteroides strains DPC 7232 and DPC 7261, Fructobacillus fructosus DPC 7237, and Fructobacillus fructosus DPC 7238, produced higher mannitol concentrations than did the positive control strain Limosilactobacillus reuteri DSM 20016 during an enzymatic screening assay. Mannitol concentrations were also examined via HPLC in 1% fructose de Man, Rogosa, and Sharpe medium (FMRS) or 1% fructose milk (FM). Among the strains, Fructobacillus fructosus DPC 7238 displayed high fructose utilization (9.27 g/L), high mannitol yield (0.99 g of mannitol/g of fructose), and greatest volumetric productivities (0.46 g/L per h) in FMRS. However, Leuconostoc mesenteroides DPC 7261 demonstrated the highest fructose utilization (8.99 g/L), mannitol yield (0.72 g of mannitol/g of fructose), and volumetric productivities (0.04 g/L per h) in FM. Storage modulus G′ (>0.1 Pa) indicated a shorter gelation time for Limosilactobacillus reuteri DSM 20016 (8.73 h), followed by F. fructosus DPC 7238 (11.57 h) and L. mesenteroides DPC 7261 (14.52 h). Our results show that fructose-rich niches can be considered important sources of fructophilic LAB strains, with the potential to be used as starter cultures or adjunct cultures for the manufacture of mannitol-enriched fermented dairy products and beverages.
Losses in crop yields due to disease need to be reduced in order to meet increasing global food demands associated with growth in the human population. There is a well recognised need to develop new environmentally-friendly control strategies to combat bacterial crop disease. Current control measures involving the use of traditional chemicals or antibiotics are losing their efficacy due to the natural development of bacterial resistance to these agents. In addition, there is an increasing awareness that their use is environmentally unfriendly. Bacteriophages, the viruses of bacteria, have received increased research interest in recent years as a realistic environmentally friendly means of controlling bacterial diseases. Their use presents a viable control measure for a number of destructive bacterial crop diseases, with some phage-based products already becoming available on the market. Phage biocontrol possesses advantages over chemical controls in that tailor-made phage cocktails can be adapted to target specific disease-causing bacteria. Unlike chemical control measures, phage mixtures can be easily adapted for bacterial resistance which may develop over time. In this review, we will examine the progress and challenges for phage-based disease biocontrol in food crops.
Listeria monocytogenes is a foodborne pathogen that is characterised by its ability to withstand mild stresses (i.e. cold, acid, salt) often encountered in food products or food processing environments. In the previous phenotypic and genotypic characterisation of a collection of L. monocytogenes strains, we have identified one strain 1381, originally obtained from EURL-lm, as acid sensitive (reduced survival at pH 2.3) and extremely acid intolerant (no growth at pH 4.9, which supports the growth of most strains). In this study, we investigated the cause of acid intolerance in strain 1381 by isolating and sequencing reversion mutants that were capable of growth at low pH (pH 4.8) to a similar extent as another strain (1380) from the same clonal complex (CC2). Whole genome sequencing showed that a truncation in mntH, which encodes a homologue of an NRAMP type Mn2+ transporter, is responsible for the acid intolerance phenotype observed in strain 1381. However, the mntH truncation alone was not sufficient to explain the acid sensitivity of strain 1381 at lethal pH values as strain 1381R1 (a mntH+ revertant) exhibited similar acid survival to its parental strain at pH 2.3. Further growth experiments demonstrated that Mn2+ (but not Fe2+, Zn2+, Cu2+, Ca2+, or Mg2+) supplementation fully rescues the growth of strain 1381 under low pH conditions, suggesting that a Mn2+ limitation is the likely cause of growth arrest in the mntH- background. Consistent with the important role of Mn2+ in the acid stress response was the finding that mntH and mntB (both encoding Mn2+ transporters) had higher transcription levels following exposure to mild acid stress (pH 5). Taken together, these results provide evidence that MntH-mediated Mn2+ uptake is essential for the growth of L. monocytogenes under low pH conditions. Moreover, since strain 1381 was recommended for conducting food challenge studies by the European Union Reference Laboratory, the use of this strain in evaluating the growth of L. monocytogenes in low pH environments where Mn2+ is scarce should be reconsidered.
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