Tenacibaculum aquimarinum sp. nov., isolated from a marine alga and seawater.

Two Gram-stain-negative, aerobic and yellow-pigmented bacterial strains, designated K20-16T and MSW2, were isolated from a marine red alga (Chondrus species) and seawater, respectively. Both strains were oxidase-positive, weakly catalase-positive and non-flagellated rods with gliding motility. Menaquinone-6 was detected as the sole isoprenoid quinone in both strains. Iso-C15:0, iso-C15:0 3-OH, iso-C15:1 G, C15:1 ω6c and summed feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c) were identified in both strains as major fatty acids. Phosphatidylethanolamine was not identified in strain K20-16T, but it was identified in strain MSW2. The genomic DNA G+C contents of strains K20-16T and MSW2 were 30.5 and 30.7 %, respectively. Strains K20-16T and MSW2 shared 99.7% 16S rRNA gene sequence similarity, 97.7% average nucleotide identity (ANI), and 80.5% digital DNA-DNA hybridization (DDH) value, indicating that they are the same species. Phylogenetic analyses based on 16S rRNA gene and 92 concatenated core protein sequences revealed that strains K20-16T and MSW2 formed a phylogenic lineage within the genus Tenacibaculum and were most closely related to Tenacibaculum todarodis LPB0136T with 98.3 and 98.0% 16S rRNA gene sequence similarities, respectively. ANI and digital DDH values between strains K20-16T and MSW2 and other type strains were less than 91.4 and 43.1 %, respectively. Based on the phenotypic, chemotaxonomic and molecular features, strains K20-16T and MSW2 represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum aquimarinum sp. nov. is proposed. The type strain is K20-16T (=KACC 22 342T=JCM 35 023T).

Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae.

We evaluated the Cre-lox and CRISPR-Cas9 systems as marker-recycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4-based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura- transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.

Colwellia maritima sp. nov. and Polaribacter marinus sp. nov., isolated from seawater.

Two Gram-stain-negative, catalase- and oxidase-positive, and aerobic bacteria, strains MSW7T and MSW13T, were isolated from seawater. Cells of strains MSW7T and MSW13T are motile and non-motile rods, respectively. Strain MSW7T optimally grew at 25 °C and pH 7.0 and in the presence of 3 % (w/v) NaCl, whereas strain MSW13T optimally grew at 25 °C and pH 6.0-7.0 and in the presence of 2 % NaCl. As the sole respiratory quinone and the major fatty acids and polar lipids, strain MSW7T contained ubiquinone-8, C16 : 0, C15 : 1 ω8c, C17 : 1 ω8c and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), and phosphatidylethanolamine and phosphatidylglycerol, respectively, whereas strain MSW13T contained menaquinone-6, C15 : 1 ω6c, iso-C15 : 0, anteiso-C15 : 0, and iso-C15 : 0 3-OH, and phosphatidylethanolamine, respectively. The DNA G+C contents of strains MSW7T and MSW13T were 37.3 and 29.9 %, respectively. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains MSW7T and MSW13T were most closely related to Colwellia echini A3T and Polaribacter atrinae WP25T with 98.8 and 98.1 % sequence similarities, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain MSW7T and C. echini A3T and between strain MSW13T and P. atrinae KACC 17473T were 73.6 and 22.6 % and 80.4 and 23.8 %, respectively. Based on phenotypic, chemotaxonomic and phylogenetic data, strains MSW7T and MSW13T represent novel species of the genera Colwellia and Polaribacter, respectively, for which the names Colwellia maritima sp. nov. and Polaribacter marinus sp. nov. are proposed, respectively. The type strains of C. maritima sp. nov. and P. marinus sp. nov. are MSW7T (=KACC 22339T=JCM 35001T) and MSW13T (=KACC 22341T=JCM 35021T), respectively.

Mec1 Modulates Interhomolog Crossover and Interplays with Tel1 at Post Double-Strand Break Stages.

During meiosis I, programmed DNA double-strand breaks (DSBs) occur to promote chromosome pairing and recombination between homologs. In Saccharomyces cerevisiae, Mec1 and Tel1, the orthologs of human ATR and ATM, respectively, regulate events upstream of the cell cycle checkpoint to initiate DNA repair. Tel1ATM and Mec1ATR are required for phosphorylating various meiotic proteins during recombination. This study aimed to investigate the role of Tel1ATM and Mec1ATR in meiotic prophase via physical analysis of recombination. Tel1ATM cooperated with Mec1ATR to mediate DSB-to-single end invasion transition, but negatively regulated DSB formation. Furthermore, Mec1ATR was required for the formation of interhomolog joint molecules from early prophase, thus establishing a recombination partner choice. Moreover, Mec1ATR specifically promoted crossover-fated DSB repair. Together, these results suggest that Tel1ATM and Mec1ATR function redundantly or independently in all post-DSB stages.

Ku complex suppresses recombination in the absence of MRX activity during budding yeast meiosis.

During meiosis, programmed double-strand breaks (DSBs) are repaired via recombination pathways that are required for faithful chromosomal segregation and genetic diversity. In meiotic progression, the non-homologous end joining (NHEJ) pathway is suppressed and instead meiotic recombination initiated by nucleolytic resection of DSB ends is the major pathway employed. This requires diverse recombinase proteins and regulatory factors involved in the formation of crossovers (COs) and non-crossovers (NCOs). In mitosis, spontaneous DSBs occurring at the G1 phase are predominantly repaired via NHEJ, mediating the joining of DNA ends. The Ku complex binds to these DSB ends, inhibiting additional DSB resection and mediating end joining with Dnl4, Lif1, and Nej1, which join the Ku complex and DSB ends. Here, we report the role of the Ku complex in DSB repair using a physical analysis of recombination in Saccharomyces cerevisiae during meiosis. We found that the Ku complex is not essential for meiotic progression, DSB formation, joint molecule formation, or CO/NCO formation during normal meiosis. Surprisingly, in the absence of the Ku complex and functional Mre11-Rad50-Xrs2 (MRX) complex, a large portion of meiotic DSBs was repaired via the recombination pathway to form COs and NCOs. Our data suggested that Ku complex prevents meiotic recombination in the elimination of MRX activity. [BMB Reports 2019; 52(10): 607-612].

The nature of meiotic chromosome dynamics and recombination in budding yeast.

During meiosis, crossing over allows for the exchange of genes between homologous chromosomes, enabling their segregation and leading to genetic variation in the resulting gametes. Spo11, a topoisomerase-like protein expressed in eukaryotes, and diverse accessory factors induce programmed double-strand breaks (DSBs) to initiate meiotic recombination during the early phase of meiosis after DNA replication. DSBs are further repaired via meiosis-specific homologous recombination. Studies on budding yeast have provided insights into meiosis and genetic recombination and have improved our understanding of higher eukaryotic systems. Cohesin, a chromosome-associated multiprotein complex, mediates sister chromatid cohesion (SCC), and is conserved from yeast to humans. Diverse cohesin subunits in budding yeast have been identified in DNA metabolic pathways, such as DNA replication, chromosome segregation, recombination, DNA repair, and gene regulation. During cell cycle, SCC is established by multiple cohesin subunits, which physically bind sister chromatids together and modulate proteins that involve in the capturing and separation of sister chromatids. Cohesin components include at least four core subunits that establish and maintain SCC: two structural maintenance chromosome subunits (Smc1 and Smc3), an α-kleisin subunit (Mcd1/Scc1 during mitosis and Rec8 during meiosis), and Scc3/Irr1 (SA1 and SA2). In addition, the cohesin-associated factors Pds5 and Rad61 regulate structural modifications and cell cyclespecific dynamics of chromatin to ensure accurate chromosome segregation. In this review, we discuss SCC and the recombination pathway, as well as the relationship between the two processes in budding yeast, and we suggest a possible conserved mechanism for meiotic chromosome dynamics from yeast to humans.

Roles of Budding Yeast Hrr25 in Recombination and Sporulation.

Hrr25, a casein kinase 1 δ/ε homolog in budding yeast, is essential to set up mono-orientation of sister kinetochores during meiosis. Hrr25 kinase activity coordinates sister chromatid cohesion via cohesin phosphorylation. Here, we investigated the prophase role of Hrr25 using the auxin-inducible degron system and by ectopic expression of Hrr25 during yeast meiosis. Hrr25 mediates nuclear division in meiosis I but does not affect DNA replication. We also found that initiation of meiotic double-strand breaks as well as joint molecule formation were normal in HRR25-deficient cells. Thus, Hrr25 is essential for termination of meiotic division but not homologous recombination.