Generation of bacteriophage-insensitive mutants of Streptococcus thermophilus using an antisense RNA CRISPR-Cas silencing approach

Predation of starter lactic acid bacteria such as Streptococcus thermophilus by bacteriophages is a persistent and costly problem in the dairy industry. CRISPR-mediated Bacteriophage Insensitive Mutants (BIMs), while straightforward to generate and verify, can quickly be overcome by mutant phages. The aim of this study was to develop a tool allowing the generation of derivatives of commercial S. thermophilus strains which are resistant to phage attack through a non-CRISPR-mediated mechanism, with the objective of generating BIMs exhibiting stable resistance against a range of isolated lytic S. thermophilus phages. To achieve this, standard BIM generation was complemented by the use of the wild-type (WT) strain which had been transformed with an antisense mRNA-generating plasmid (targeting a crucial CRISPR-associated [cas] gene], in order to facilitate the generation of non-CRISPR-mediated BIMs. Phage sensitivity assays suggest that non-CRISPR-mediated BIMs exhibit some advantages compared to CRISPR-mediated BIMs derived from the same strain.Importance: The outlined approach reveals the presence of a powerful host-imposed barrier for phage infection in S. thermophilus. Considering the detrimental economic consequences of phage infection in the dairy processing environment, the developed methodology has widespread applications, particularly where other methods may not be practical or effective in obtaining robust, phage-tolerant S. thermophilus starter strains

Genome sequences of eight prophages isolated from Lactococcus lactis dairy strains

P335 group phages represent the most divergent phage group infecting dairy Lactococcus lactis strains and have significant implications for the dairy processing industry. Here, we report the complete genome sequences of eight lactococcal prophages chemically induced from industrial lactococcal strains that propagate lytically on one of two laboratory strain

Detecting Lactococcus lactis prophages by Mitomycin C-mediated induction coupled to flow cytometry analysis

Most analyzed Lactococcus lactis strains are predicted to harbor one or more prophage genomes within their chromosome; however, the true extent of the inducibility and functionality of such prophages cannot easily be deduced from sequence analysis alone. Chemical treatment of lysogenic strains with Mitomycin C is known to cause induction of temperate phages, though it is not always easy to clearly identify a lysogenic strain or to measure the number of released phage particles. Here, we report the application of flow cytometry as a reliable tool for the detection and enumeration of released lactococcal prophages using the green dye SYTO-9

Genetic and functional characterisation of the lactococcal P335 phage-host interactions

Background: Despite continuous research efforts, bacterio(phages) infecting Lactococcus lactis starter strains persist as a major threat to dairy fermentations. The lactococcal P335 phages, which are currently classified into four sub-groups (I-IV), are the second most frequently isolated phage group in an industrial dairy context. Results: The current work describes the isolation and comparative genomic analysis of 17 novel P335 group phages. Detailed analysis of the genomic region of P335 phages encoding the so-called “baseplate”, which includes the receptor binding protein (RBP) was combined with a functional characterization of the RBP of sub-group III and IV phages. Additionally, calcium-dependence assays revealed a specific requirement for calcium by sub-group IV phages while host range analysis highlighted a higher number of strains with CWPS type A (11 of 39 strains) are infected by the P335 phages assessed in this study than those with a C (five strains), B (three of 39 strains) or unknown (one of 39 strains) CWPS type. Conclusions: These analyses revealed significant divergence among RBP sequences, apparently reflecting their unique interactions with the host and particularly for strains with a type A CWPS. The implications of the genomic architecture of lactococcal P335 phages on serving as a general model for Siphoviridae phages are discussed

Peningkatan Aktivitas Belajar Mahasiswa melalui Lesson Study pada Mata Kuliah Anatomi dan Morfologi Tumbuhan

Tujuan yang ingin dicapai pada kegiatan lesson study ini adalah untuk meningkatkan aktivitas belajar mahasiswa di Program Studi Pendidikan Biologi pada mata kuliah Anatomi dan Morfologi Tumbuhan. Hasil akhir yang diharapkan adalah semua kompetensi dasar dan standar kompetensi bisa dicapai. Aktivitas belajar yang diharapkan meningkat terutama adalah (1) kemampuan berpikir kritis, (2) kemampuan berkomunikasi lisan, (3) kemampuan bekerja sama dalam tim, (4) kedisiplinan. Lesson Study dilakukan dengan menerapkan problem solving dalam perkuliahan dan dilakukan dalam 4 kali siklus yang masing-masing terdiri dari kegiatan Plan, Do dan See. Plan dilakukan oleh dosen model dan observer untuk menganalisis kebutuhan dan permasalahan yang dihadapi dalam perkuliahan dan mempersiapkan semua instrumen dan perangkat kuliah yang diyakini mampu membelajarkan mahasiswa secara efektif serta membangkitkan partisipasi aktif mahasiswa dalam pembelajaran. Produk dalam kegiatan plan adalah jadwal pelaksanaan plan, do dan see, RPP, materi, media, dan alat evaluasi. Kegiatan selanjutnya adalah kegiatan open class, yaitu kegiatan tatap muka di kelas yang diampu oleh dosen model dan dimonitor keterlaksanaannya oleh para observer. Setelah itu dosen model dan para observer melakukan refleksi berdasarkan open class yang baru saja dilakukan, mendiskusikan masalah dan cara penanggulangannya agar siklus selanjutnya lebih baik pencapaiannya. Berdasarkan hasil pelaksanaan lesson study selama 4 siklus didapatkan bahwa penggunaan model problem solving melalui lesson study pada pembelajaran Anatomi dan Morfologi Tumbuhan dapat meningkatkan aktivitas belajar mahasiswa pada setiap tahapan siklus, yang ditunjukkan dengan meningkatnya (1) kemampuan berpikir kritis, (2) kemampuan berkomunikasi lisan, (3) kemampuan bekerja sama dalam tim, dan (4) kedisiplinan. Selain itu juga terdapat peningkatan kualitas pembelajaran yang dilakukan oleh dosen, baik dalam persiapan pembelajaran maupun keterampilan melaksanakan pembelajaran. Walaupun demikian, lesson study ini masih perlu dilanjutkan dan ditingkatkan agar pembelajaran yang berkualitas tetap terjaga

Model Sistem Multi Agen Linear Dengan Formasi Segitiga

In this paper, a linear model of multi agent movement in equilateral triangle formation is considered. The agents have initial and final state in triangular formation. Along the motion, all agents can not move far away and collide. The agents are steered from initial position to final position in fixed time. For this goal, optimal control with Pontryagin Maximum Principle is applied and the classic difficulty in the optimal control problem is appear. To solve the classic difficulty above, the steepest descent method is used

A cell wall-associated polysaccharide is required for bacteriophage adsorption to the Streptococcus thermophilus cell surface

Streptococcus thermophilus strain ST64987 was exposed to a member of a recently discovered group of S. thermophilus phages (the 987 phage group), generating phage-insensitive mutants, which were then characterized phenotypically and genomically. Decreased phage adsorption was observed in selected bacteriophage-insensitive mutants, and was partnered with a sedimenting phenotype and increased cell chain length or aggregation. Whole genome sequencing of several bacteriophage-insensitive mutants identified mutations located in a gene cluster presumed to be responsible for cell wall polysaccharide production in this strain. Analysis of cell surface-associated glycans by methylation and NMR spectroscopy revealed a complex branched rhamno-polysaccharide in both ST64987 and phage-insensitive mutant BIM3. In addition, a second cell wall-associated polysaccharide of ST64987, composed of hexasaccharide branched repeating units containing galactose and glucose, was absent in the cell wall of mutant BIM3. Genetic complementation of three phage-resistant mutants was shown to restore the carbohydrate and phage resistance profiles of the wild-type strain, establishing the role of this gene cluster in cell wall polysaccharide production and phage adsorption and, thus, infection

Global Survey and Genome Exploration of Bacteriophages Infecting the Lactic Acid Bacterium Streptococcus thermophilus

Despite the persistent and costly problem caused by (bacterio)phage predation of Streptococcus thermophilus in dairy plants, DNA sequence information relating to these phages remains limited. Genome sequencing is necessary to better understand the diversity and proliferative strategies of virulent phages. In this report, whole genome sequences of 40 distinct bacteriophages infecting S. thermophilus were analyzed for general characteristics, genomic structure and novel features. The bacteriophage genomes display a high degree of conservation within defined groupings, particularly across the structural modules. Supporting this observation, four novel members of a recently discovered third group of S. thermophilus phages (termed the 5093 group) were found to be conserved relative to both phage 5093 and to each other. Replication modules of S. thermophilus phages generally fall within two main groups, while such phage genomes typically encode one putative transcriptional regulator. Such features are indicative of widespread functional synteny across genetically distinct phage groups. Phage genomes also display nucleotide divergence between groups, and between individual phages of the same group (within replication modules and at the 3′ end of the lysis module)—through various insertions and/or deletions. A previously described multiplex PCR phage detection system was updated to reflect current knowledge on S. thermophilus phages. Furthermore, the structural protein complement as well as the antireceptor (responsible for the initial attachment of the phage to the host cell) of a representative of the 5093 group was defined. Our data more than triples the currently available genomic information on S. thermophilus phages, being of significant value to the dairy industry, where genetic knowledge of lytic phages is crucial for phage detection and monitoring purposes. In particular, the updated PCR detection methodology for S. thermophilus phages is highly useful in monitoring particular phage group(s) present in a given whey sample. Studies of this nature therefore not only provide information on the prevalence and associated threat of known S. thermophilus phages, but may also uncover newly emerging and genomically distinct phages infecting this dairy starter bacterium