RESUMO
Lactic Acid Bacteria (LAB) have a long history of safe use in milk fermentation and are generally recognized as health-promoting microorganisms when present in fermented foods. LAB are also important components of the human intestinal microbiota and are widely used as probiotics. Considering their safe and health-beneficial properties, LAB are considered appropriate vehicles that can be genetically modified for food, industrial and pharmaceutical applications. Here, this review describes (1) the potential opportunities for application of genetically modified LAB strains in dairy fermentation and (2) the various genomic modification tools for LAB strains, such as random mutagenesis, adaptive laboratory evolution, conjugation, homologous recombination, recombineering, and CRISPR (clustered regularly interspaced short palindromic repeat)- Cas (CRISPR-associated protein) based genome engineering. Lastly, this review also discusses the potential future developments of these genomic modification technologies and their applications in dairy fermentations.
RESUMO
Fructophilic Lactic Acid Bacteria (FLAB), Fructobacillus fructosus DPC7238 and pseudofructophilic Leuconostoc mesenteroides DPC7261 and non-FLAB Limosilactobacillus reuteri DSM20016 strains were studied for their growth and morphological evolution as a function of increased fructose concentrations (0, 25, and 50% w/v) in the media. A comparison of the genomics of these strains was carried out to relate observed changes and understand fructose-rich adaptations. The viability of FLAB strains were reduced by approx. 50% at a 50% fructose concentration, while the Limosilactobacillus reuteri strain was reduced to approx. 98%. Electron microscopy demonstrated that FLAB strain, Fructobacillus. fructosus and pseudofructophilic Leuc. mesenteroides, were intact but expanded in the presence of high fructose in the medium. Limosilactobacillus reuteri, on the other hand, ruptured as a result of excessive elongation, resulting in the formation of cell debris when the medium contained more than 25% (w/v) fructose. This was entirely and quantitatively corroborated by three-dimensional data obtained by scanning several single cells using an atomic force microscope. The damage caused the bacterial envelope to elongate lengthwise, thus increasing width size and lower height. The cell surface became comparatively smoother at 25% fructose while rougher at 50% fructose, irrespective of the strains. Although Fructobacillus fructosus was highly fructose tolerant and maintained topological integrity, it had a comparatively smaller genome than pseudofructophilic Leuc. mesenteroides. Further, COG analysis identified lower but effective numbers of genes in fructose metabolism and transport of Fructobacillus fructosus, essentially needed for adaptability in fructose-rich niches.
Assuntos
Lactobacillales , Lactobacillales/genética , Lactobacillales/metabolismo , Frutose/metabolismo , Genômica , Ácido Láctico/metabolismoRESUMO
To understand the molecular mechanisms that contribute to the stress responses of the important foodborne pathogen Listeria monocytogenes, we collected 139 strains (meat, n = 25; dairy, n = 10; vegetable, n = 8; seafood, n = 14; mixed food, n = 4; and food processing environments, n = 78), mostly isolated in Ireland, and subjected them to whole-genome sequencing. These strains were compared to 25 Irish clinical isolates and 4 well-studied reference strains. Core genome and pan-genome analysis confirmed a highly clonal and deeply branched population structure. Multilocus sequence typing showed that this collection contained a diverse range of strains from L. monocytogenes lineages I and II. Several groups of isolates with highly similar genome content were traced to single or multiple food business operators, providing evidence of strain persistence or prevalence, respectively. Phenotypic screening assays for tolerance to salt stress and resistance to acid stress revealed variants within several clonal complexes that were phenotypically distinct. Five of these phenotypic outliers were found to carry mutations in the sigB operon, which encodes the stress-inducible sigma factor sigma B. Transcriptional analysis confirmed that three of the strains that carried mutations in sigB, rsbV, or rsbU had reduced SigB activity, as predicted. These strains exhibited increased tolerance to salt stress and displayed decreased resistance to low pH stress. Overall, this study shows that loss-of-function mutations in the sigB operon are comparatively common in field isolates, probably reflecting the cost of the general stress response to reproductive fitness in this pathogen. IMPORTANCE The bacterial foodborne pathogen Listeria monocytogenes frequently contaminates various categories of food products and is able to cause life-threatening infections when ingested by humans. Thus, it is important to control the growth of this bacterium in food by understanding the mechanisms that allow its proliferation under suboptimal conditions. In this study, intraspecies heterogeneity in stress response was observed across a collection consisting of mainly Irish L. monocytogenes isolates. Through comparisons of genome sequence and phenotypes observed, we identified three strains with impairment of the general stress response regulator SigB. Two of these strains are used widely in food challenge studies for evaluating the growth potential of L. monocytogenes. Given that loss of SigB function is associated with atypical phenotypic properties, the use of these strains in food challenge studies should be re-evaluated.
Assuntos
Proteínas de Bactérias , Listeria monocytogenes , Fator sigma , Proteínas de Bactérias/genética , Microbiologia de Alimentos , Listeria monocytogenes/genética , Fenótipo , Filogenia , Fator sigma/genéticaRESUMO
A collection of Listeria monocytogenes isolates from various food products, food processing environments and clinical sources (n = 153) were evaluated for their tolerance to acetic, lactic and propionic acids. A large variation in tolerance was observed amongst isolates under mildly acidic conditions (pH 5.3) for acetic (5-20 mM undissociated acid) and propionic acid (2-10 mM undissociated acid) but there was less variation for lactic acid (3-6 mM undissociated acid). Analysis of the isolate genome sequences for a complement of genes previously shown to have a role in acid tolerance revealed that thiT, gadT2, gadD2 and gadD3 genes were linked to higher acetic acid tolerance (P < 0.05) while lisRK was linked to higher tolerance to propionic acid (P = 1 × 10-11). An absence of plasmid genes was also linked with isolates showing higher tolerance for all acids. Scoary GWAS analysis revealed that a total of 333, 207, and 333 genes were associated with acid tolerance for acetic, lactic, and propionic acid, respectively (P < 0.05). However, the p-value adjusted with Bonferroni's method for multiple comparisons did not reveal any significant associations. Isolates were grouped into clonal complexes (CC) using Multi Locus Sequence Typing (MLST) and MIC values for the three acids were determined for representative strains. One complex, CC18, showed significantly higher (P ≤ 0.05) acetic and propionic acid MIC values than other groups, whereas only CC7 type isolates revealed significantly higher (P ≤ 0.001) lactic acid MIC values. The results demonstrate that MLST typing could be linked to acid tolerance phenotypic traits which is important in predicting the behaviour of L. monocytogenes in food products.
Assuntos
Listeria monocytogenes , Manipulação de Alimentos , Microbiologia de Alimentos , Genótipo , Listeria monocytogenes/genética , Tipagem de Sequências MultilocusRESUMO
The genus Lactobacillus has represented an extremely large and diverse collection of bacteria that populate a wide range of habitats, and which may have industrial applications. Researchers have grappled with the immense genetic, metabolic, and ecological diversity within the genus Lactobacillus for many years. As a result, the taxonomy of lactobacilli has been extensively revised, incorporating new genus names for many lactobacilli based on their characteristics including genomic similarities. As a result, many lactobacilli traditionally associated with dairy products now have new genus names and are grouped into new clades or clusters of species. In this review, we examine how the taxonomic restructuring of the genus Lactobacillus will affect the dairy industry and discuss lactobacilli associated with dairy production, processing, and those that confer possible health benefits when delivered by dairy products.
Assuntos
Laticínios , Lactobacillus , Animais , Bactérias , Laticínios/microbiologia , Indústria de Laticínios , Genômica , Lactobacillus/metabolismoRESUMO
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.
Assuntos
Lactobacillales/metabolismo , Manitol/metabolismo , Leite/metabolismo , Animais , Produtos Fermentados do Leite , Frutose/metabolismo , Géis/metabolismo , Lactobacillales/classificação , Lactobacillales/isolamento & purificação , Lactobacillus/isolamento & purificação , Lactococcus/isolamento & purificação , Leuconostoc/isolamento & purificação , Leuconostocaceae , RNA Ribossômico 16SRESUMO
Flavor is one of the most important attributes of any fermented dairy product. Dairy consumers are known to be willing to experiment with different flavors; thus, many companies producing fermented dairy products have looked at culture manipulation as a tool for flavor diversification. The development of flavor is a complex process, originating from a combination of microbiological, biochemical, and technological aspects. A key driver of flavor is the enzymatic activities of the deliberately inoculated starter cultures, in addition to the environmental or "nonstarter" microbiota. The contribution of microbial metabolism to flavor development in fermented dairy products has been exploited for thousands of years, but the availability of the whole genome sequences of the bacteria and yeasts involved in the fermentation process and the possibilities now offered by next-generation sequencing and downstream "omics" technologies is stimulating a more knowledge-based approach to the selection of desirable cultures for flavor development. By linking genomic traits to phenotypic outputs, it is now possible to mine the metabolic diversity of starter cultures, analyze the metabolic routes to flavor compound formation, identify those strains with flavor-forming potential, and select them for possible commercial application. This approach also allows for the identification of species and strains not previously considered as potential flavor-formers, the blending of strains with complementary metabolic pathways, and the potential improvement of key technological characteristics in existing strains, strains that are at the core of the dairy industry. An in-depth knowledge of the metabolic pathways of individual strains and their interactions in mixed culture fermentations can allow starter blends to be custom-made to suit industry needs. Applying this knowledge to starter culture research programs is enabling research and development scientists to develop superior starters, expand flavor profiles, and potentially develop new products for future market expansion.
Assuntos
Bactérias/genética , Bactérias/metabolismo , Produtos Fermentados do Leite/microbiologia , Aromatizantes/metabolismo , Fungos/genética , Microbiota , Animais , Bactérias/isolamento & purificação , Produtos Fermentados do Leite/análise , Fermentação , Aromatizantes/química , Fungos/isolamento & purificação , Fungos/metabolismo , Genômica , Humanos , PaladarRESUMO
BACKGROUND: Strains of Lactobacillus paracasei are present in many diverse environments, including dairy and plant materials and the intestinal tracts of humans and animals. Their adaptation to various niches is correlated to intra-species diversity at the genomic and metabolic level. In this study, we compared the genome sequences of three L. paracasei strains isolated from mature Cheddar cheeses, two of which (DPC4206 and DPC4536) shared the same genomic fingerprint by PFGE, but demonstrated varying metabolic capabilities. RESULTS: Genome sizes varied from 2.9 Mbp for DPC2071, to 3.09 Mbp for DPC4206 and 3.08 Mpb for DPC4536. The presence of plasmids was a distinguishing feature between the strains with strain DPC2071 possessing an unusually high number of plasmids (up to 11), while DPC4206 had one plasmid and DPC4536 harboured no plasmids. Each of the strains possessed specific genes not present in the other two analysed strains. The three strains differed in their abundance of sugar-converting genes, and in the types of sugars that could be used as energy sources. Genes involved in the metabolism of sugars not usually connected with the dairy niche, such as myo-inositol and pullulan were also detected, but strains did not utilise these sugars. The genetic content of the three strains differed in regard to specific genes for arginine and sulfur-containing amino acid metabolism and genes contributing to resistance to heavy metal ions. In addition, variability in the presence of phage remnants and phage protection systems was evident. CONCLUSIONS: The findings presented in this study confirm a considerable level of heterogeneity of Lactobacillus paracasei strains, even between strains isolated from the same niche.
Assuntos
Queijo/microbiologia , Variação Genética , Genômica/métodos , Lacticaseibacillus paracasei/genética , Lacticaseibacillus paracasei/metabolismo , Metabolômica/métodos , Animais , Queijo/análise , DNA Bacteriano/genética , Genoma Bacteriano , Lacticaseibacillus paracasei/classificação , Lacticaseibacillus paracasei/isolamento & purificação , FilogeniaRESUMO
The Gram-positive genus Macrococcus is composed of eight species that are evolutionarily closely related to species of the Staphylococcus genus. In contrast to Staphylococcus species, species of Macrococcus are generally regarded to be avirulent in their animal hosts. Recent reports on Macrococcus have focused on the presence of novel methicillin resistance genes in Macrococcus caseolyticus and Macrococcus canis, with the discovery of the first plasmid-encoded methicillin resistance gene in clinical Staphylococcus aureus of probable macrococcal origin generating further interest in these organisms. Furthermore, M. caseolyticus has been associated with flavor development in certain fermented foods and its potential as a food bio-preservative has been documented. The potential application of these organisms in food seems at odds with the emerging information regarding antibiotic resistance and is prompting further examination of the potential safety issues associated with such strains, given the European Food Safety Authority framework for the safety evaluation of microorganisms in the food chain. A comprehensive understanding of the genus would also contribute to understanding the evolution of staphylococci in terms of its acquisition of antibiotic resistance and pathogenic potential. In this review, we discuss the current knowledge on Macrococcus with regard to their phenotypic capabilities, genetic diversity, and evolutionary history with Staphylococcus. Comparative genomics of the sequenced Macrococcus species will be discussed, providing insight into their unique metabolic features and the genetic structures carrying methicillin resistance. An in-depth understanding of these antibiotic resistance determinants can open the possibilities for devising better preventative strategies for an unpredictable future.
Assuntos
Evolução Biológica , Microbiologia de Alimentos , Infecções por Bactérias Gram-Positivas/veterinária , Resistência a Meticilina , Staphylococcaceae/genética , Staphylococcaceae/fisiologia , Animais , Inocuidade dos Alimentos , Genes Bacterianos , Variação Genética , Infecções por Bactérias Gram-Positivas/microbiologia , Redes e Vias Metabólicas/genética , Staphylococcaceae/efeitos dos fármacos , Staphylococcaceae/isolamento & purificaçãoRESUMO
Campylobacter phage vB_CjeM_Los1 was recently isolated from a slaughterhouse in the Republic of Ireland using the host Campylobacter jejuni subsp. jejuni PT14, and full-genome sequencing and annotation were performed. The genome was found to be 134,073 bp in length and to contain 169 predicted open reading frames. Transmission electron microscopy images of vB_CjeM_Los1 revealed that it belongs to the family Myoviridae, with tail fibres observed in both extended and folded conformations, as seen in T4. The genome size and morphology of vB_CjeM_Los1 suggest that it belongs to the genus Cp8virus, and seven other Campylobacter phages with similar size characteristics have also been fully sequenced. In this work, comparative studies were performed in relation to genomic rearrangements and conservation within each of the eight genomes. None of the eight genomes were found to have undergone internal rearrangements, and their sequences retained more than 98% identity with one another despite the widespread geographical distribution of each phage. Whole-genome phylogenetics were also performed, and clades were shown to be representative of the differing number of tRNAs present in each phage. This may be an indication of lineages within the genus, despite their striking homology.
Assuntos
Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Genoma Viral , Myoviridae/genética , Matadouros , Animais , Bacteriófagos/classificação , Bacteriófagos/ultraestrutura , Campylobacter/virologia , Genômica , Irlanda , Microscopia Eletrônica de Transmissão , Myoviridae/classificação , Myoviridae/isolamento & purificação , Fases de Leitura Aberta , Filogenia , Aves Domésticas/virologia , Proteínas Virais/genéticaRESUMO
The widespread dissemination of species of the lactic acid bacteria (LAB) group in different environments testifies to their extraordinary niche adaptability. Members of the LAB are present on grass and other plant material, in dairy products, on human skin, and in the gastrointestinal and reproductive tracts. The selective pressure imparted by these specific environments is a key driver in the genomic diversity observed between strains of the same species deriving from distinct habitats. Strains that are exploited in the dairy industry for the production of fermented dairy products are often referred to as "domesticated" strains. These strains, which initially may have occupied a nondairy niche, have become specialized for growth in the milk environment. In fact, comparative genome analysis of multiple LAB species and strains has revealed a central trend in LAB evolution: the loss of ancestral genes and metabolic simplification toward adaptation to nutritionally rich environments. In contrast, "environmental" strains, or those from raw milk, plants, and animals, exhibit diverse metabolic capabilities and lifestyle characteristics compared with their domesticated counterparts. Because of the limited number of established dairy strains used in fermented food production today, demand is increasing for novel strains, with concerted efforts to mine the microbiota of natural environments for strains of technological interest. Many studies have concentrated on uncovering the genomic and metabolic potential of these organisms, facilitating comparative genome analysis of strains from diverse environments and providing insight into the natural diversity of the LAB, a group of organisms that is at the core of the dairy industry. The natural biodiversity that exists in these environments may be exploited in dairy fermentations to expand flavor profiles, to produce natural "clean label" ingredients, or to develop safer products.
Assuntos
Biodiversidade , Queijo/microbiologia , Lactococcus lactis , Animais , Humanos , Lactococcus lactis/genética , Lactococcus lactis/isolamento & purificação , Lactococcus lactis/metabolismo , Leite/microbiologiaRESUMO
The Lactobacillus genus represents the largest and most diverse genera of all the lactic acid bacteria (LAB), encompassing species with applications in industrial, biotechnological and medical fields. The increasing number of available Lactobacillus genome sequences has allowed understanding of genetic and metabolic potential of this LAB group. Pangenome and core genome studies are available for numerous species, demonstrating the plasticity of the Lactobacillus genomes and providing the evidence of niche adaptability. Advancements in the application of lactobacilli in the dairy industry lie in exploring the genetic background of their commercially important characteristics, such as flavour development potential or resistance to the phage attack. The integration of available genomic and metabolomic data through the generation of genome scale metabolic models has enabled the development of computational models that predict the behaviour of organisms under specific conditions and present a route to metabolic engineering. Lactobacilli are recognised as potential cell factories, confirmed by the successful production of many compounds. In this review, we discuss the current knowledge of genomics, metabolomics and metabolic engineering of the prevalent Lactobacillus species associated with the production of fermented dairy foods. In-depth understanding of their characteristics opens the possibilities for their future knowledge-based applications.
Assuntos
Laticínios/microbiologia , Genoma Bacteriano , Lactobacillus/genética , Lactobacillus/metabolismo , Engenharia Metabólica , Metaboloma , Microbiologia de Alimentos , Transferência Genética Horizontal , Genômica , Lactobacillus/classificação , Lactobacillus/fisiologia , Metabolômica , FilogeniaRESUMO
Cheese flavor development is directly connected to the metabolic activity of microorganisms used during its manufacture, and the selection of metabolically diverse strains represents a potential tool for the production of cheese with novel and distinct flavor characteristics. Strains of Lactobacillus have been proven to promote the development of important cheese flavor compounds. As cheese production and ripening are long-lasting and expensive, model systems have been developed with the purpose of rapidly screening lactic acid bacteria for their flavor potential. The biodiversity of 10 strains of the Lactobacillus casei group was evaluated in 2 model systems and their volatile profiles were determined by gas chromatography-mass spectrometry. In model system 1, which represented a mixture of free AA, inoculated cells did not grow. In total, 66 compounds considered as flavor contributors were successfully identified, most of which were aldehydes, acids, and alcohols produced via AA metabolism by selected strains. Three strains (DPC2071, DPC3990, and DPC4206) had the most diverse metabolic capacities in model system 1. In model system 2, which was based on processed cheese curd, inoculated cells increased in numbers over incubation time. A total of 47 compounds were identified, and they originated not only from proteolysis, but also from glycolytic and lipolytic processes. Tested strains produced ketones, acids, and esters. Although strains produced different abundances of volatiles, diversity was less evident in model system 2, and only one strain (DPC4206) was distinguished from the others. Strains identified as the most dissimilar in both of the model systems could be more useful for cheese flavor diversification.
Assuntos
Queijo/microbiologia , Indústria de Laticínios/métodos , Aromatizantes/metabolismo , Lacticaseibacillus casei/metabolismo , Paladar , Animais , Aromatizantes/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Lactobacillus , Lacticaseibacillus casei/classificaçãoRESUMO
Endolysins (lysins) are bacteriophage-encoded enzymes that have evolved to degrade specific bonds within the bacterial cell wall. These enzymes represent a novel class of antibacterial agents against infectious pathogens, especially in light of multidrug-resistant bacteria, which have made antibiotic therapy increasingly redundant. Lysins have been used successfully to eliminate/control bacterial pathogens in various anatomical locations in mouse and other animal models. Engineering tactics have also been successfully applied to improve lysin function. This review discusses the structure and function of lysins. It highlights protein-engineering tactics utilised to improve lysin activity. It also reviews the applications of lysins towards food biopreservation, therapeutics, biofilm elimination and diagnostics.
RESUMO
The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on August 28-September 1, 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting.
Assuntos
Química Farmacêutica/métodos , Proteínas/química , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Epigênese Genética , Relação Estrutura-Atividade , Biologia de SistemasRESUMO
Bacteriophages and their derivatives are continuously gaining impetus as viable alternative therapeutic agents to control harmful multidrug-resistant bacterial pathogens, particularly in the food industry. The reduced efficacy of conventional antibiotics has resulted in a quest to find novel alternatives in the war against infectious disease. This study describes the full-genome sequence of Cronobacter phage vB_CsaP_Ss1, with subsequent cloning and expression of its endolysin, capable of hydrolysing Gram-negative peptidoglycan. Cronobacter phage vB_CsaP_Ss1 is composed of 42â205 bp of dsDNA with a G+C content of 46.1âmol%. A total of 57 ORFs were identified of which 18 could be assigned a putative function based on similarity to characterized proteins. The genome of Cronobacter phage vB_CsaP_Ss1 showed little similarity to any other bacteriophage genomes available in the database and thus was considered unique. In addition, functional analysis of the predicted endolysin (LysSs1) was also investigated. Zymographic experiments demonstrated the hydrolytic activity of LysSs1 against Gram-negative peptidoglycan, and this endolysin thus represents a novel candidate with potential for use against Gram-negative pathogens.
Assuntos
Bacteriófagos/genética , Parede Celular/efeitos dos fármacos , Cronobacter/virologia , Endopeptidases/genética , Endopeptidases/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Peptidoglicano/metabolismo , Composição de Bases , Parede Celular/metabolismo , DNA Viral/química , DNA Viral/genética , Genoma Viral , Hidrólise , Dados de Sequência Molecular , Fases de Leitura Aberta , Controle Biológico de Vetores/métodos , Análise de Sequência de DNARESUMO
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 â¼14%, 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.
Assuntos
Variação Genética , Lactococcus lactis/classificação , Lactococcus lactis/genética , Poaceae/microbiologia , Verduras/microbiologia , Animais , Bovinos , Genoma Bacteriano , Genótipo , Lactococcus lactis/isolamento & purificação , Lactococcus lactis/fisiologia , Leite/microbiologia , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Fenótipo , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Lactococcus lactis is an organism of substantial economic importance, used extensively in the production of fermented foods and widely held to have evolved from plant strains. The domestication of this organism to the milk environment is associated with genome reduction and gene decay, and the acquisition of specific genes involved in protein and lactose utilisation by horizontal gene transfer. In recent years, numerous studies have focused on uncovering the physiology and molecular biology of lactococcal strains from the wider environment for exploitation in the dairy industry. This in turn has facilitated comparative genome analysis of lactococci from different environments and provided insight into the natural phenotypic and genetic diversity of L. lactis. This diversity may be exploited in dairy fermentations to develop products with improved quality and sensory attributes. In this review, we discuss the classification of L. lactis and the problems that arise with phenotype/genotype designation. We also discuss the adaptation of non-dairy lactococci to milk, the traits associated with this adaptation and the potential application of non-dairy lactococci to dairy fermentations.
Assuntos
Fermentação , Lactococcus lactis/fisiologia , Leite/microbiologia , Plantas/microbiologia , Animais , Laticínios/microbiologia , Meio Ambiente , Transferência Genética Horizontal , Variação Genética , Genoma Bacteriano , Genótipo , Lactococcus lactis/classificação , Lactococcus lactis/genética , Lactococcus lactis/isolamento & purificação , Fenótipo , FilogeniaRESUMO
Listeria monocytogenes is a pathogenic bacterium that can inhabit a diverse range of environmental niches. This is largely attributed to the high proportion of carbohydrate-specific phosphotransferase system (PTS) genes in its genome. Carbohydrates can be assimilated as sources of energy but additionally they can serve as niche-specific cues for L. monocytogenes to shape its global gene expression, in order to cope with anticipated stresses. To examine carbon source utilization among wild L. monocytogenes isolates and to understand underlying molecular mechanisms, a diverse collection of L. monocytogenes strains (n = 168) with whole genome sequence (WGS) data available was screened for the ability to grow in chemically defined media with different carbon sources. The majority of the strains grew in glucose, mannose, fructose, cellobiose, glycerol, trehalose, and sucrose. Maltose, lactose, and rhamnose supported slower growth while ribose did not support any growth. In contrast to other strains, strain1386, which belonged to clonal complex 5 (CC5), was unable to grow on trehalose as a sole carbon source. WGS data revealed that it carried a substitution (N352K) in a putative PTS EIIBC trehalose transporter, TreB, while this asparagine residue is conserved in other strains in this collection. Spontaneous mutants of strain 1386 that could grow in trehalose were found to harbour a reversion of the substitution in TreB. These results provide genetic evidence that TreB is responsible for trehalose uptake and that the N352 residue is essential for TreB activity. Moreover, reversion mutants also restored other unusual phenotypes that strain 1386 displayed, i.e. altered colony morphology, impaired biofilm development, and reduced acid resistance. Transcriptional analysis at stationary phase with buffered BHI media revealed that trehalose metabolism positively influences the transcription of genes encoding amino acid-based acid resistance mechanisms. In summary, our results demonstrated that N352 is key to the function of the sole trehalose transporter TreB in L. monocytogenes and suggest that trehalose metabolism alters physiology to favour biofilm development and acid stress resistance. Moreover, since strain 1386 is among the strains recommended by the European Union Reference Laboratory for conducting food challenge studies in order to determine whether or not L. monocytogenes can grow in food, these findings have important implications for food safety.
Assuntos
Listeria monocytogenes , Trealose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboidratos , Proteínas de Membrana Transportadoras , BiofilmesRESUMO
Stomach acid provides a significant innate barrier to the entry of the food-borne pathogen Listeria monocytogenes into the human gastrointestinal tract. A key determinant of acid resistance in this bacterium is the conserved glutamate decarboxylase system, GadD2 (encoded by the gadT2D2 operon), which helps to maintain the intracellular pH during exposure to gastric acid. In this study, we identified a premature stop codon in a gene located immediately downstream of the gadT2D2 operon that was highly linked to an acid-sensitive phenotype. When this open reading frame was restored through homologous recombination, an acid-resistant phenotype was restored. Through a series of genetic, transcriptomic, and survival experiments, we established that this gene, which we designated gadR, encodes a transcriptional regulator of the gadT2D2 operon. GadR belongs to the RofA family of regulators, primarily found in streptococci, where they are involved in regulating virulence. The data further showed that gadR plays a critical role in the development of acid resistance in response to mild acid exposure, a response that is known as the adaptive acid tolerance response (ATR). A deletion analysis of the gadT2D2 promoter region identified two 18-bp palindromic sequences that are required for the GadR-mediated induction of gadT2D2, suggesting that they act as binding sites for GadR. Overall, this study uncovers a new RofA-like regulator of acid resistance in L. monocytogenes, which plays a significant role in both growth phase-dependent acid resistance and ATR and accounts for previously observed strain-to-strain differences in survival at low pH.IMPORTANCEThe ability to survive the acidic conditions found in the stomach is crucial for the food-borne pathogen Listeria monocytogenes to gain access to the mammalian gastrointestinal tract. Little is currently known about how acid resistance is regulated in this pathogen and why this trait is highly variable between strains. Here, we used comparative genomics to identify a novel RofA-family transcriptional regulator, GadR, that controls the development of acid resistance. The RofA family of regulators was previously found only in a small group of bacterial pathogens, including streptococci, where they regulate virulence properties. We show that gadR encodes the dominant regulator of acid resistance in L. monocytogenes and that its sequence variability accounts for previously observed differences between strains in this trait. Together, these findings significantly advance our understanding of how this important pathogen copes with acid stress and suggest a potential molecular target to aid its control in the food chain.