'Lactobacillus fermentum' 3872 genome sequencing and analysis

In recent years, there has been a rise in antimicrobial-resistant bacteria caused by overdependence on, and misuse of, antibiotics. This has led to an increase in research for identifying alternatives to combat pathogens. One promising means of combating pathogenic bacteria, particularly for those residing in the gastrointestinal tract (GIT), is the use of probiotics. This thesis focuses on a potential probiotic strain Lactobacillus fermentum 3872, the genome sequence of which was circularised during the study, identifying genes that may contribute to probiotic activity. Several genes involved in GIT survival, such as acid symporters were discovered, along with genes that encode adhesion proteins such as those involved in mucus, fibronectin and collagen binding. The genes mentioned above may contribute to L. fermentum 3872 survivability within the GIT and have an antagonistic effect on enteric pathogens via competitive exclusion. Other interesting genes identified in L. fermentum 3872 were potentially involved in bacterial aggregation, exopolysaccharide and vitamin synthesis, along with four prophage encoding regions. Genes that encode a class III bacteriocin was also identified. An additional gene encoding a collagen binding protein (CBP) of a newly discovered plasmid pLF3872, was recognised. The chromosomal sequence also had a partial CBP encoding gene. pLF3872 has a toxin-antitoxin gene pair that ensures stable maintenance of the plasmid, along with conjugation-related genes. Functional analysis of the recombinant CBP via ELISA experiments found that the protein had the ability to bind to collagen I, a protein present on the epithelial lining of cells of the GIT. ELISA experiments also demonstrated that a common gastrointestinal pathogen, Campylobacter jejuni, can bind to collagen I in a concentration-dependent manner. In addition, mass spectrometry analysis identified that C. jejuni strains 11168H and 81-176 may utilise flagellar components (FlaA and FlaB) for adhesion. Furthermore, C. jejuni 11168H and 81-176 binding to collagen I was inhibited in the presence of either L. fermentum 3872 or CBP, thus reducing C. jejuni adherence via competitive exclusion. Using an in vitro assay, it was also demonstrated that L. fermentum 3872 cell-free supernatant could inhibit the growth of C. jejuni, due to the acidic environment brought about by L. fermentum 3872. During the completion of the genome sequence of L. fermentum 3872, comparison of various sequence assembly techniques which focused on the quality of the genome assembly was conducted. The results showed that further extension of the genome sequence during sequence assembly may lead to assembly errors when over-relying on a commonly-used sequence quality indicator, referred to as read mapping. It is suggested that care must also be taken when using long read technology to complete the genome sequence of a bacteria, as this may result in nucleotide sequence redundancies

Survival of Campylobacter jejuni 11168H in Acanthamoebae castellanii Provides Mechanistic Insight into Host Pathogen Interactions

Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis worldwide but is rarely transferred between human hosts. Although a recognized microaerophile, the majority of C. jejuni are incapable of growing in an aerobic environment. The persistence and transmission of this pathogen outside its warm-blooded avian and mammalian hosts is poorly understood. Acanthamoebae species are predatory protists and form an important ecological niche with several bacterial species. Here, we investigate the interaction of C. jejuni 11168H and Acanthamoebae castellanii at the single-cell level. We observe that a subpopulation of C. jejuni cells can resist killing by A. castellanii, and non-digested bacteria are exocytosed into the environment where they can persist. In addition, we observe that A. castellanii can harbor C. jejuni 11168H even upon encystment. Transcriptome analyses of C. jejuni interactions revealed similar survival mechanisms when infecting both A. castellanii and warm-blooded hosts. In particular, nitrosative stress defense mechanisms and flagellum function are important as confirmed by mutational analyses of C. jejuni 11168H. This study describes a new host–pathogen interaction for C. jejuni and confirms that amoebae are transient hosts for the persistence, adaptability, and potential transmission of C. jejuni

Whole Genome Sequence of Dermacoccus abyssi MT1.1 Isolated from the Challenger Deep of the Mariana Trench Reveals Phenazine Biosynthesis Locus and Environmental Adaptation Factors

Dermacoccus abyssi strain MT1.1T is a piezotolerant actinobacterium that was isolated from Mariana Trench sediment collected at a depth of 10898 m. The organism was found to produce ten dermacozines (A‒J) that belonged to a new phenazine family and which displayed various biological activities such as radical scavenging and cytotoxicity. Here, we report on the isolation and identification of a new dermacozine compound, dermacozine M, the chemical structure of which was determined using 1D and 2D-NMR, and high resolution MS. A whole genome sequence of the strain contained six secondary metabolite-biosynthetic gene clusters (BGCs), including one responsible for the biosynthesis of a family of phenazine compounds. A pathway leading to the biosynthesis of dermacozines is proposed. Bioinformatic analyses of key stress-related genes provide an insight into how the organism adapted to the environmental conditions that prevail in the deep-sea

Biotechnological and ecological potential of 'Micromonospora provocatoris' sp. nov., a gifted strain isolated from the Challenger Deep of the Mariana Trench

A Micromonospora strain, isolate MT25T, was recovered from a sediment collected from the Challenger Deep of the Mariana Trench using a selective isolation procedure. The isolate produced two major metabolites, n-acetylglutaminyl glutamine amide and desferrioxamine B, the chemical structures of which were determined using 1D and 2D-NMR, including 1H-15N HSQC and 1H-15N HMBC 2D-NMR, as well as high resolution MS. A whole genome sequence of the strain showed the presence of ten natural product-biosynthetic gene clusters, including one responsible for the biosynthesis of desferrioxamine B. Whilst 16S rRNA gene sequence analyses showed that the isolate was most closely related to the type strain of Micromonospora chalcea, a whole genome sequence analysis revealed it to be most closely related to Micromonospora tulbaghiae 45142T. The two strains were distinguished using a combination of genomic and phenotypic features. Based on these data, it is proposed that strain MT25T (NCIMB 15245T, TISTR 2834T) be classified as Micromonospora provocatoris sp. nov. Analysis of the genome sequence of strain MT25T (genome size 6.1 Mbp) revealed genes predicted to responsible for its adaptation to extreme environmental conditions that prevail in deep-sea sediments

Potential use of ' Lactobacillus fermentum ' 3872 as an anti-campylobacter agent

Due to the rise of multidrug resistant form of pathogenic microorganisms, alternative intervention tools are in urgent need. One option is to employ beneficial (probiotic) bacteria, which could compete with pathogens for host cell attachment cites and elicit antibacterial activity. Lactobacillus fermentum strain 3872, which revealed outstanding probiotic activity, is a good candidate for such studies. Genome sequencing of this strain revealed a novel plasmid (pLF3872) containing a gene, encoding a collagen-binding proteins (CBP). The plasmid was not found in any other strains of the species. The chromosomal genome sequence of this strain (2.3 Mb) was found to contain other genes potentially contributing to its beneficial effects, such as those encoding a mucus binding protein and other adhesins, as well as a bacteriocin-encoding gene not found in other sequenced genomes of these bacteria. ELISA-based attachment experiments revealed competition of L. fermentum 3872 with Campylobacter jejuni strain 11168H for binding to collagen I, which is a ubiquitous structure making up the gastrointestinal tract. The results suggest that L. fermentum 3872 can potentially be used for competitive exclusion of Campylobacter jejuni, which is the most important gastrointestinal pathogen

Additional file 1: Table S1. of Potential probiotic-associated traits revealed from completed high quality genome sequence of Lactobacillus fermentum 3872

Associated MIGS record. (DOC 72 kb

Campylobacter jejuni interactions with Acanthamoeba castellanii

To investigate the interaction of intra-amoebal C. jejuni with the transient host A. castellanii. We then performed gene expression profiling analysis using data obtained from RNA-seq of control and intra-amoebal C. jejuni

Investigation into the efficiency of diverse N-linking oligosaccharyltransferases for glycoengineering using a standardised cell-free assay.

Publication status: PublishedThe application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity

Phenotypic and genotypic characterization of Campylobacter coli isolates from the Vietnamese poultry production network; a pilot study

Introduction Changing farming practices and the associated increase in the use of antibiotics are amongst the main drivers shaping the global increase of Campylobacter infections. The effects farming practices have on Campylobacter species, need to be studied at the global scale, particularly in emerging middle-income countries, where the demand for low-cost poultry meat is rising. While Campylobacter jejuni causes the majority of poultry associated diarrhoea, Campylobacter coli causes a significant amount of disease but are relatively understudied. In this study we characterised seven C. coli strains isolated from chicken faeces and chicken meat in Thai Nguyen province, Vietnam. Methods Whole Genome Sequencing and phenotypic assays (growth, motility, antimicrobial resistance testing, virulence assay) were performed to reveal the genetic relatedness and pathophysiological characteristics of the isolates. Results All isolates were resistant to ciprofloxacin and nalidixic acid but susceptible to phenicols. Three were resistant to macrolides azithromycin and erythromycin. Six isolates were classified as multi-drug resistant. All isolates had similar growth rates in laboratory culture media, while five were hyper-motile. Lethality towards a tractable host-model system, larvae of the greater wax moth Galleria mellonella, often used to determine Campylobacter virulence, was demonstrated for the first time for C. coli. Discussion Multilocus sequence typing data identified five ST’s all within the C. coli ST-828 clonal complex and were previously reported in North American (ST-829), European (ST-1586), and Asia (ST-5511) from patients suffering from gastroenteritis, emphasising the global spread of these strains. This work highlights the importance of further research into this understudied global threat

Whole genome sequencing of four bacterial strains from South Shetland Trench revealing biosynthetic and environmental adaptation gene clusters

Whole genome sequences of four bacterial strains Dietzia maris SST1, Pseudomonas zhaodongensis SST2, Pseudomonas sp. SST3 and Halomonas sulfidaeris SST4, recovered from the South Shetland Trench sediment in Antarctica were analyzed using Ion Torrent sequencing technology. The respective sizes of their genomes (3.88, 4.99, 5.60 and 4.25 Mb) and GC contents (70.0, 60.3, 59.9 and 53.8%) are in agreement with these values of other strains of the species. The bacterial strains displayed promising antimicrobial activity against a number of pathogenic bacterial and fungal species. Whole genomes have been assembled and biosynthetic gene clusters (BGCs) have been identified using the antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) web platform. Comparative analysis of the genome sequences revealed that the strains host abundant BGCs encoding for terpenes, siderophores, arylpolyene, bacteriocins, and lassopeptides. Furthermore, the key stress-related genes were identified and their distribution provided an insight into how these isolates adapt to key marine environmental conditions. This comprehensive study is a contribution to understanding the nature of life on the deep-sea environments