Application of wheat flour (Triticum aestivum) on spore density and sporulation efficiency of Bacillus megaterium isolated from Litopenaeus vannamei gastrointestinal tract

Mahariawan IMD, Kusuma WE, Yuniarti A, Beltran MAG, Hariati AM. 2021. Application of wheat flour (Triticum aestivum) on spore density and sporulation efficiency of Bacillus megaterium isolated from Litopenaeus vannamei gastrointestinal tract. Biodiversitas 22: 3709-3715. Bacillus megaterium is frequently used in fish farming, such as white shrimp (Litopenaeus vannamei) pond, which can produce spores with high stability in its implementation. Currently, spore production still requires the availability of high-cost carbon sources. The objective of this research was to evaluate the effect of different wheat flour doses on spore density and sporulation efficiency of B. megaterium BM1. In flasks, 50 mL of each test medium was treated with different doses of wheat (10, 20, 30 and 40 g. L-1, respectively) and glucose was used as a control. Each treatment was inoculated with B. megaterium BM1 (2.6 x 108 CFU. mL-1) and incubated in a shaker incubator (120 rpm) at 37 °C for 120 hours. The results showed that the highest vegetative cell concentration (17 x 108 CFU. mL-1), growth rate (0.8 hour-1) and spore (14.7 x 108 spores. mL-1) were found in the wheat flour dose of 30 g. L-1. Furthermore, the highest sporulation efficiency was achieved at 20 g. L-1 of wheat (91.30%) and germination should be done at a dose less than 40 g. L-1. The size of the spores was 1.35-1.39 µm. Thus, 30 g. L-1 of wheat flour is a potential dose to produce spore for probiotic candidates.

Cdc14 activates autophagy to regulate spindle pole body dynamics during meiosis

Autophagy, a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues, is regulated by multiple signaling pathways that oversee cell survival, growth, and proliferation. In budding yeast, autophagy plays an essential role in meiotic exit, although the molecular mechanisms underlying its regulation and cargo selection remain unknown. In this study, we found that autophagy is maintained during meiosis and is upregulated at anaphase I and anaphase II. In addition, we found that cells with higher levels of autophagy during meiosis I and II completed meiosis faster, and that genetically activated autophagy machinery increased sporulation efficiency. Strikingly, our data revealed that Cdc14, a highly conserved phosphatase that counteracts Cdc28 (CDK1), is a meiosis-specific autophagy regulator. At anaphase I and anaphase II, Cdc14 was activated and released from the nucleolus into the cytoplasm, where it dephosphorylated Atg13 to stimulate Atg1 kinase activity and thus autophagy. Importantly, the meiosis-specific spindle pole body (SPB, the yeast centrosome) component (Spo74) was sensitized to autophagy-mediated degradation at anaphase II, upon its dephosphorylation by Cdc14. Together, our findings reveal a meiosis-tailored mechanism of Cdc14 that spatiotemporally guides meiotic autophagy activity to control SPB dynamics.

Elevated Sporulation Efficiency in Fission Yeast Schizosaccharomyces japonicus Strains Isolated from Drosophila

The fission yeast Schizosaccharomyces japonicus, comprising S. japonicus var. japonicus and S. japonicus var. versatilis varieties, has unique characteristics such as striking hyphal growth not seen in other Schizosaccharomyces species; however, information on its diversity and evolution, in particular mating and sporulation, remains limited. Here we compared the growth and mating phenotypes of 17 wild strains of S. japonicus, including eight S. japonicus var. japonicus strains newly isolated from an insect (Drosophila). Unlike existing wild strains isolated from fruits/plants, the strains isolated from Drosophila sporulated at high frequency even under nitrogen-abundant conditions. In addition, one of the strains from Drosophila was stained by iodine vapor, although the type strain of S. japonicus var. japonicus is not stained. Sequence analysis further showed that the nucleotide and amino acid sequences of pheromone-related genes have diversified among the eight strains from Drosophila, suggesting crossing between S. japonicus cells of different genetic backgrounds occurs frequently in this insect. Much of yeast ecology remains unclear, but our findings suggest that insects such as Drosophila might be a good niche for mating and sporulation, and will provide a basis for the understanding of sporulation mechanisms via signal transduction, as well as the ecology and evolution of yeast.

Transcriptional coupling and repair of 8-OxoG activate a RecA-dependent checkpoint that controls the onset of sporulation in Bacillus subtilis

During sporulation Bacillus subtilis Mfd couples transcription to nucleotide excision repair (NER) to eliminate DNA distorting lesions. Here, we report a significant decline in sporulation following Mfd disruption, which was manifested in the absence of external DNA-damage suggesting that spontaneous lesions activate the function of Mfd for an efficient sporogenesis. Accordingly, a dramatic decline in sporulation efficiency took place in a B. subtilis strain lacking Mfd and the repair/prevention guanine oxidized (GO) system (hereafter, the ∆GO system), composed by YtkD, MutM and MutY. Furthermore, the simultaneous absence of Mfd and the GO system, (i) sensitized sporulating cells to H2O2, and (ii) elicited spontaneous and oxygen radical-induced rifampin-resistance (Rifr) mutagenesis. Epifluorescence (EF), confocal and transmission electron (TEM) microscopy analyses, showed a decreased ability of ∆GO ∆mfd strain to sporulate and to develop the typical morphologies of sporulating cells. Remarkably, disruption of sda, sirA and disA partially, restored the sporulation efficiency of the strain deficient for Mfd and the ∆GO system; complete restoration occurred in the RecA− background. Overall, our results unveil a novel Mfd mechanism of transcription-coupled-repair (TCR) elicited by 8-OxoG which converges in the activation of a RecA-dependent checkpoint event that control the onset of sporulation in B. subtilis.

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt “bookmark” sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and “bookmark”-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique “watermark” through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

Fission Yeast Methylenetetrahydrofolate Reductase Ensures Mitotic and Meiotic Chromosome Segregation Fidelity

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway, and its loss of function through polymorphisms is often associated with human conditions, including cancer, congenital heart disease, and Down syndrome. MTHFR is also required in the maintenance of heterochromatin, a crucial determinant of genomic stability and precise chromosomal segregation. Here, we characterize the function of a fission yeast gene met11+, which encodes a protein that is highly homologous to the mammalian MTHFR. We show that, although met11+ is not essential for viability, its disruption increases chromosome missegregation and destabilizes constitutive heterochromatic regions at pericentromeric, sub-telomeric and ribosomal DNA (rDNA) loci. Transcriptional silencing at these sites were disrupted, which is accompanied by the reduction in enrichment of histone H3 lysine 9 dimethylation (H3K9me2) and binding of the heterochromatin protein 1 (HP1)-like Swi6. The met11 null mutant also dominantly disrupts meiotic fidelity, as displayed by reduced sporulation efficiency and defects in proper partitioning of the genetic material during meiosis. Interestingly, the faithful execution of these meiotic processes is synergistically ensured by cooperation among Met11, Rec8, a meiosis-specific cohesin protein, and the shugoshin protein Sgo1, which protects Rec8 from untimely cleavage. Overall, our results suggest a key role for Met11 in maintaining pericentromeric heterochromatin for precise genetic inheritance during mitosis and meiosis.

Isolation and characterization of Bacillus spp. strains as potential probiotics for poultry

Probiotics have become one of the potential solutions to global restriction on antibiotic use in food animal production. Bacillus species have been attractive probiotics partially due to their long-term stability during storage. In this study, 200 endospore-forming bacteria isolates were recovered from sourdough and the gastrointestinal tract (GIT) of young broiler chicks. Based on the production of a series of exoenzymes and survivability under stress conditions similar to those in the poultry GIT, 42 isolates were selected and identified by 16S rRNA gene sequencing. Seven strains with a profile of high enzymatic activities were further evaluated for sporulation efficiency, biofilm formation, compatibility among themselves (Bacillus spp.), and antagonistic effects against three bacteria pathogenic to poultry and humans: Enterococcus cecorum, Salmonella enterica, and Shiga-toxin-producing Escherichia coli. The strains from sourdough were identified as Bacillus amyloliquefaciens whereas the ones from the chicks’ GIT were Bacillus subtilis. These strains demonstrated remarkable potential as probiotics for poultry.

Petrobactin protects against oxidative stress and enhances sporulation efficiency inBacillus anthracisSterne

Bacillus anthracisis a gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is the only such system known to be required for vegetative growth ofB. anthracisin iron-depleted conditions,e.g., in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficientin vitrosporulation ofB. anthracisrequires petrobactin, that the petrobactin biosynthesis operon (asbA-F) is induced prior to sporulation, and that petrobactin itself is associated with spores. Petrobactin is also required for both oxidative stress protection during late stage growth and wild-type levels of sporulation in sporulation medium. When considered with the petrobactin-dependent sporulation in bovine blood also described in this work, these effects onin vitrogrowth and sporulation suggest that petrobactin is required forB. anthracistransmission via the spore during natural infections in addition to its key functions during active anthrax infections.ImportanceBacillus anthraciscauses the disease anthrax, which is transmitted via its dormant, spore phase. However, converting from bacilli to spore is a complex, energetically costly process that requires many nutrients including iron.B. anthracisrequires the siderophore petrobactin to scavenge iron from host environments. We show that in the Sterne strain, petrobactin is required also for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high iron sporulation medium but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.

STRESS RESPONSES IN PIGMENTED BACILLUS PUMILUS SF14

Microbial producers of carotenoids belongs to a various species of unicellular algae, filamentous fungi and several bacteria. A recent report has shown that up to 15% of aerobic spore-formers identified from soil samples are pigmented and in many cases the pigments are carotenoids. Pigmented spore-forming Bacillus was obtained from culture maintained in the microbial culture collection of Department of Structural Functional Biol. University of Naples Federico II and partially characterized their pigments. A classical mutagenesis approach has been used to obtain mutant strains producing altered pigments or no pigments. Our results suggest that pigmentation in spore’s represent an additional, and may be alternative protection strategy against oxidative stress. A mutants (SF214-M1, SF214-M2, SF214-M3 and SF214-M4) of Bacillus pumilus strain SF214 producing a carotenoids water soluble-pigment were obtained after treatment with the mutagenic agent N-Methyl-N-nitroso-N'-nitroguanidine (NTG). Several microbiological and biochemical properties of these 4 strains were analyzed and the results were differences between wild type and other four mutants in producing pigments, color changing, sporulation, cannot produce spores after mutation and sporulation efficiency was constant with color development.