When spheroplasts of the yeast Saccharomyces cerevisiae are cultured in liquid medium containing osmotic stabilizer, they undergo nuclear division and growth without cell division, resulting in the formation of giant spheroplasts with multinuclei. In this study, we report a simple method for the culture and stable maintenance of giant spheroplasts. The selection of culture media and cell concentration was found to be important for the growth and maintenance of giant spheroplasts. Among the conditions that we tested, static culture in a synthetic Burkholder’s medium in 96-well U-bottomed culture plates was most effective. Under appropriate conditions, we could maintain giant spheroplasts for more than 6 days without proliferation of whole cells or marked lysis. The average diameter of spheroplasts can vary from 16 to 53μm, depending on their initial concentration.
The genetic and functional diversity of Bacillus and Bacillus-derived genera was analyzed in soil samples collected from three different fields near Century Paper Mill, Lal Kuan, Uttarakhand, India. Two had been subjected to concentrated and diluted effluent irrigation for the past 25 years and were designated as a concentrated effluent irrigated field (CEIF) and a dilute effluent irrigated field (DEIF), respectively. The field irrigated with fresh water was designated as a water irrigated field (WIF). Increase in pH, Na and Zn content and decrease in Fe content was observed due to effluent irrigation. The population count of Bacillus and Bacillus-derived was maximum in DEIF followed by WIF and CEIF. Variations in plant growth-promoting traits and extracellular enzymes were observed among the isolates from the three different field soils. Based on the amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes, all the selected 104 isolates were clustered into 14 groups. The sequencing of the representative isolates revealed that the majority belonged to the genus Bacillus, while three isolates belonged to Paenibacillus, Lysinibacillus and Orthinibacillus. There were a few species like Orthinibacillus contaminans, B. oleronius, B. safensis, B. methylotrophicus, B. stratosphericus, B. aryabhattai, B. asahii and B. bataviensis that were prevalent only in DEIF and CEIF but not in WIF field soil. The diversity index parameters like the Shannon Index, indices of species richness and species evenness based on biochemical profiling and ARDRA profiling revealed that Bacillaceae members in the fresh water irrigated field were diverse.
Sour congee is a popular food in the western regions of Inner Mongolia in China. It has a complex microbial population, which contributes to its unique flavor and functional properties. In this study, we chose 28 sour congee samples that were collected from different areas in Inner Mongolia for analysis of the microbial community of lactic acid bacteria (LAB) by classical biochemical tests, 16S rRNA gene sequencing, multiplex PCR assay of recA gene and restriction fragment length polymorphism (RFLP) analysis of the tuf gene (encoding elongation factor Tu). The results revealed that all the isolates were identified as Lactobacillus (L.) paracasei (38 strains), L. fermentum (28 strains), L. plantarum (7 strains), L. brevis (4 strains), L. reuteri (2 strains), L. amylolyticus (1 strain), Enterococcus (E.) faecalis (3 strains), E. italicus (2 strains) or Lactococcus lactis subsp. lactis (1 strain). The predominant LAB were L. casei and L. fermentum in sour congee samples. The diversity of LAB derived from sour congee could offer useful information for further research on sour congee, and the results demonstrated that the combination of tuf gene and RFLP patterns can be considered as a useful tool for differentiation of the L. casei group.
A spontaneous non-pathogenic variant (Cong:1-2) derived from Fusarium oxysporum f. sp. conglutinans (Cong: 1-1), a causal agent of cabbage yellows, carries biocontrol activity for cabbage yellows. We found a GMC oxidoreductase (ODX1) among the proteins expressed much more in Cong:1-2 than Cong:1-1 by 2D-DIGE comparison. GMC oxidoreductases have been reported to be involved in biocontrol activity of several plant pathogenic fungi. The gene encoding ODX1 in Cong:1-2 was cloned, and targeted disruption of the gene in Cong:1-2 did not affect its biocontrol activity, suggesting that GMC oxidoreductase is dispensable for biocontrol activity in the fungal biocontrol agent.
In Escherichia coli (E. coli), most DNA damage-inducible (din) genes belong to the LexA regulon, whose products are related to functions such as DNA repair and induced mutagenesis. The E. coli K-12 cells have about 30 operons that are known to be members of the LexA regulon. LexA acts as a transcriptional repressor of these unlinked genes by binding to the specific DNA sequences located within the promoter regions. We developed a genetic screening method to isolate LexA dependent promoters. By using an applied whole-genome shotgun method with a lac-operon system, we isolated promoter candidates of din genes from the E. coli O157:H7 genome. We found that transcriptional repression from most of these promoters was dependent on lexA and purified LexA protein bound directly to the DNA fragments carrying them. Finally, we identified 16 and 5 promoters that regulated expression of previously known and novel LexA dependent genes, respectively. In addition to them, we also identified 2 antisense promoters which were considered to regulate expression of antisense RNAs for mRNAs of the ecs1779 and ecs2988 genes. All newly identified promoter regions contained DNA sequences similar to the consensus LexA binding sequence.
Four strains of yeasts isolated in Thailand and Taiwan were found to represent four distinct novel species of the ascomycetous anamorphic yeast genus Candida. These strains are located in the Clavispora-Metschnikowia clade in a phylogenetic tree based on the D1/D2 domain sequences of the large subunit rRNA genes. Together with Candida picinguabensis and Candida saopaulonensis, the four novel species constitute a well-separated subclade from other species of the Clavispora-Metschnikowia clade. Three species from Thailand are described as Candida bambusicola sp. nov. (type strain, ST-50T = BCC 7750T = NBRC 106734T = CBS 11723T), Candida nongkhaiensis sp. nov. (type strain, ST-95T = BCC 8331T = NBRC 105874T =CBS 11724T) and Candida succicola sp. nov. (type strain, ST-631T = BCC 15314T = NBRC 106736T = CBS 11726T), respectively, and the species from Taiwan is described as Candida touchengensis sp. nov. (type strain, SY4S03T = NBRC 102647T = BCRC 23097T = CBS 10585T).