Extraction and Analysis of Plasmid DNA from Listeria monocytogenes.

A plasmid preparation is a method used to extract and purify plasmid DNA. Methods developed to purify plasmid DNA from bacteria generally involve harvesting and alkaline lysis of the bacteria, precipitation of chromosomal DNA and protein, followed by purification of the plasmid DNA. Here, we describe the mini-preparation of plasmid DNA by a rapid small-scale method, adapted for Listeria monocytogenes. The quality of plasmid DNA isolated using this method is sufficient for analytical purposes but may be upscaled for further downstream analysis. Electrophoretic separation of the resultant lysate allows conclusions to be made on the presence, number, copy number, and size of the plasmids in the analyzed bacterial strains.

Things Are Getting Hairy: Enterobacteria Bacteriophage vB_PcaM_CBB.

Enterobacteria phage vB_PcaM_CBB is a "jumbo" phage belonging to the family Myoviridae. It possesses highly atypical whisker-like structures along the length of its contractile tail. It has a broad host range with the capability of infecting species of the genera Erwinia, Pectobacterium, and Cronobacter. With a genome of 355,922 bp, excluding a predicted terminal repeat of 22,456 bp, phage CBB is the third largest phage sequenced to date. Its genome was predicted to encode 554 ORFs with 33 tRNAs. Based on prediction and proteome analysis of the virions, 29% of its predicted ORFs could be functionally assigned. Protein comparison shows that CBB shares between 33-38% of its proteins with Cronobacter phage GAP32, coliphages PBECO4 and 121Q as well as Klebsiella phage vB_KleM_Rak2. This work presents a detailed and comparative analysis of vB_PcaM_CBB of a highly atypical jumbo myoviridae phage, contributing to a better understanding of phage diversity and biology.

Draft Genome Sequences of Listeria monocytogenes Serotype 4b Strains 944 and 2993 and Serotype 1/2c Strains 198 and 2932.

Listeria monocytogenes is a foodborne pathogen and the causative agent of listeriosis among humans and animals. The draft genome sequences of L. monocytogenes serotype 4b strains 944 and 2993 and serotype 1/2c strains 198 and 2932 are reported here.

Draft Genome Sequence of Lactobacillus casei DPC6800, an Isolate with the Potential to Diversify Flavor in Cheese.

Lactobacillus casei is a nonstarter lactic acid bacterium commonly present in various types of cheeses. It is believed that strains of this species have a significant impact on the development of cheese flavor. The draft genome sequence of L. casei DPC6800, isolated from a semi-hard Dutch cheese, is reported.

Comparative Genomic Analysis of Two Serotype 1/2b Listeria monocytogenes Isolates from Analogous Environmental Niches Demonstrates the Influence of Hypervariable Hotspots in Defining Pathogenesis.

The vast majority of clinical human listeriosis cases are caused by serotype 1/2a, 1/2b, 1/2c, and 4b isolates of Listeria monocytogenes. The ability of L. monocytogenes to establish a systemic listeriosis infection within a host organism relies on a combination of genes that are involved in cell recognition, internalization, evasion of host defenses, and in vitro survival and growth. Recently, whole genome sequencing and comparative genomic analysis have proven to be powerful tools for the identification of these virulence-associated genes in L. monocytogenes. In this study, two serotype 1/2b strains of L. monocytogenes with analogous isolation sources, but differing infection abilities, were subjected to comparative genomic analysis. The results from this comparison highlight the importance of accessory genes (genes that are not part of the conserved core genome) in L. monocytogenes pathogenesis. In addition, a number of factors, which may account for the perceived inability of one of the strains to establish a systemic infection within its host, have been identified. These factors include the notable absence of the Listeria pathogenicity island 3 and the stress survival islet, of which the latter has been demonstrated to enhance the survival ability of L. monocytogenes during its passage through the host intestinal tract, leading to a higher infection rate. The findings from this research demonstrate the influence of hypervariable hotspots in defining the physiological characteristics of a L. monocytogenes strain and indicate that the emergence of a non-pathogenic isolate of L. monocytogenes may result from a cumulative loss of functionality rather than by a single isolated genetic event.

A tail of two phages: genomic and functional analysis of Listeria monocytogenes phages vB_LmoS_188 and vB_LmoS_293 reveal the receptor-binding proteins involved in host specificity.

The physical characteristics of bacteriophages establish them as viable candidates for downstream development of pathogen detection assays and biocontrol measures. To utilize phages for such purposes, a detailed knowledge of their host interaction mechanisms is a prerequisite. There is currently a wealth of knowledge available concerning Gram-negative phage-host interaction, but little by comparison for Gram-positive phages and Listeria phages in particular. In this research, the lytic spectrum of two recently isolated Listeria monocytogenes phages (vB_LmoS_188 and vB_LmoS_293) was determined, and the genomic basis for their observed serotype 4b/4e host-specificity was investigated using comparative genomics. The late tail genes of these phages were identified to be highly conserved when compared to other serovar 4-specific Listeria phages. Spontaneous mutants of each of these phages with broadened host specificities were generated. Their late tail gene sequences were compared with their wild-type counterparts resulting in the putative identification of the products of ORF 19 of vB_LmoS_188 and ORF 20 of vB_LmoS_293 as the receptor binding proteins of these phages. The research findings also indicate that conserved baseplate architectures and host interaction mechanisms exist for Listeria siphoviruses with differing host-specificities, and further contribute to the current knowledge of phage-host interactions with regard to Listeria phages.

Complete Genome Sequence of Listeria monocytogenes Strain DPC6895, a Serotype 1/2b Isolate from Bovine Raw Milk.

Listeria monocytogenes is a foodborne pathogen and is the causative agent of listeriosis among humans and animals. The draft genome sequence of L. monocytogenes DPC6895, a serotype 1/2b strain isolated from the raw milk of a cow with subclinical bovine mastitis, is reported.

Complete Genome Sequences of vB_LmoS_188 and vB_LmoS_293, Two Bacteriophages with Specificity for Listeria monocytogenes Strains of Serotypes 4b and 4e.

Listeria monocytogenes is responsible for the rare disease listeriosis, which is associated with the consumption of contaminated food products. We report here the complete genome sequences of vB_LmoS_188 and vB_LmoS_293, phages isolated from environmental sources and that have host specificity for L. monocytogenes strains of the 4b and 4e serotypes.

Evaluation of Lactococcus lactis Isolates from Nondairy Sources with Potential Dairy Applications Reveals Extensive Phenotype-Genotype Disparity and Implications for a Revised Species.

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.

Complete Genome Sequence of vB_EcoM_112, a T-Even-Type Bacteriophage Specific for Escherichia coli O157:H7.

Bacteriophage vB_EcoM_112 (formerly e11/2) is an Escherichia coli phage with specificity for the O157:H7 serotype. The vB_EcoM_112 genome sequence shares high degrees of similarity with the phage T4 genome sequence.

Extraction and analysis of plasmid DNA from Listeria monocytogenes.

A plasmid preparation is a method used to extract and purify plasmid DNA. Methods developed to purify plasmid DNA from bacteria generally involve harvesting and alkaline lysis of the bacteria and precipitation of chromosomal DNA and protein, followed by purification of the plasmid DNA. Here, we describe the mini-preparation of plasmid DNA by a rapid small-scale method, adapted for Listeria monocytogenes. The quality of plasmid DNA isolated using this method is sufficient for analytical purposes but may be upscaled for further downstream analysis. Electrophoretic separation of the resultant lysate allows conclusions to be made on the presence, number, copy number, and size of the plasmids in the analyzed bacterial strains.

Transcriptome analysis of Listeria monocytogenes exposed to biocide stress reveals a multi-system response involving cell wall synthesis, sugar uptake, and motility.

Listeria monocytogenes is a virulent food-borne pathogen most often associated with the consumption of "ready-to-eat" foods. The organism is a common contaminant of food processing plants where it may persist for extended periods of time. A commonly used approach for the control of Listeria monocytogenes in the processing environment is the application of biocides such as quaternary ammonium compounds. In this study, the transcriptomic response of a persistent strain of L. monocytogenes (strain 6179) on exposure to a sub-lethal concentration of the quaternary ammonium compound benzethonium chloride (BZT) was assessed. Using RNA-Seq, gene expression levels were quantified by sequencing the transcriptome of L. monocytogenes 6179 in the presence (4 ppm) and absence of BZT, and mapping each data set to the sequenced genome of strain 6179. Hundreds of differentially expressed genes were identified, and subsequent analysis suggested that many biological processes such as peptidoglycan biosynthesis, bacterial chemotaxis and motility, and carbohydrate uptake, were involved in the response of L. monocyotogenes to the presence of BZT. The information generated in this study further contributes to our understanding of the response of bacteria to environmental stress. In addition, this study demonstrates the importance of using the bacterium's own genome as a reference when analysing RNA-Seq data.