The Journal of General and Applied Microbiology Vol. 63(2017) No. 3

Strains of the bacterium, Corynebacterium glutamicum, are widely used for the industrial production of L-glutamic acid and various other substances. C. glutamicum ssp. lactofermentum AJ 1511, formerly classified as Brevibacterium lactofermentum, and the closely related C. glutamicum ATCC 13032 have been used as industrial strains for more than 50 years. We determined the whole genome sequence of C. glutamicum AJ 1511 and performed genome-wide comparative analysis with C. glutamicum ATCC 13032 to determine strain-specific genetic differences. This analysis revealed that the genomes of the two industrial strains are highly similar despite the phenotypic differences between the two strains. Both strains harbored unique genes but gene transpositions or inversions were not observed. The largest unique region, a 220-kb AT-rich region located between 1.78 and 2.00 Mb position in C. glutamicum ATCC 13032 genome, was missing in the genome of C. glutamicum AJ 1511. The next two largest unique regions were present in C. glutamicum AJ 1511. The first region (413–484 kb position) contains several predicted transport proteins, enzymes involved in sugar metabolism, and transposases. The second region (1.47–1.50 Mb position) encodes restriction modification systems. A gene predicted to encode NADH-dependent glutamate dehydrogenase, which is involved in L-glutamate biosynthesis, is present in C. glutamicum AJ 1511. Strain-specific genes identified in this study are likely to govern phenotypes unique to each strain.

Anodes are crucial in determining the electricity recovery of microbial fuel cells (MFCs). In this study, graphene oxide (GO) was used as an anodic material for electricity recovery from artificial dialysis wastewater (ADWW). Anaerobic incubation of ADWW with GO for 21 days produced a hydrogel complex containing embedded microbial cells and microbially reduced GO (rGO). The rGO complex recovered 540 to 810 μA/cm³ of catalytic current from ADWW after 10 days of electrochemical cultivation at 200 mV (vs. Ag/AgCl), which was approximately thirty times higher than that recovered from graphite felt (GF), a representative anode in MFCs. High-throughput sequencing analysis of prokaryotic 16S rRNA genes revealed a predominance of the Geobacter genus (35% of all prokaryotic sequences identified), particularly in the rGO complex after 20 days of polarization. The superior electricity recovery of the rGO complex was attributable to enhanced direct electron transfer via a well-developed biofilm, while indirect electron transfer via an electron mediator occurred in culture using GF.

Numerous strains of Aspergillus oryzae are industrially used for Japanese traditional fermentation and for the production of enzymes and heterologous proteins. In A. oryzae, deletion of the ku70 or ligD genes involved in non-homologous end joining (NHEJ) has allowed high gene targeting efficiency. However, this strategy has been mainly applied under the genetic background of the A. oryzae wild strain RIB40, and it would be laborious to delete the NHEJ genes in many A. oryzae industrial strains, probably due to their low gene targeting efficiency. In the present study, we generated ligD mutants from the A. oryzae industrial strains by employing the CRISPR/Cas9 system, which we previously developed as a genome editing method. Uridine/uracil auxotrophic strains were generated by deletion of the pyrG gene, which was subsequently used as a selective marker. We examined the gene targeting efficiency with the ecdR gene, of which deletion was reported to induce sclerotia formation under the genetic background of the strain RIB40. As expected, the deletion efficiencies were high, around 60~80%, in the ligD mutants of industrial strains. Intriguingly, the effects of the ecdR deletion on sclerotia formation varied depending on the strains, and we found sclerotia-like structures under the background of the industrial strains, which have never been reported to form sclerotia. The present study demonstrates that introducing ligD mutation by genome editing is an effective method allowing high gene targeting efficiency in A. oryzae industrial strains.

Flammulina velutipes is a well-known edible mushroom cultivated all over the world. However, because of the low transformation frequency, the expensive instruments required, and the complicated, time-consuming procedures necessary, there is insufficient genetic research on F. velutipes. In this study, we report a liposome-mediated transformation (LMT) system for the genetic transformation of F. velutipes. Using the LMT system, we obtained 82 ± 4 stable F. velutipes transformants per 10⁵ protoplasts, which is a clear increase in transformation frequency compared to the other methods used. We were able to detect the expression of an EGFP reporter gene in the F. velutipes transformants using fluorescence imaging assays. Furthermore, we used this method to transfer the laccase gene into F. velutipes and found that the transcriptional level and enzymatic activity increased in these transformants. Mitotic stability analysis showed that all of the selected transformants remained mitotically stable, even after five successive rounds of sub-culturing. These results demonstrate a new transgenic approach that will facilitate F. velutipes research.

The bacterial diversity associated with biofilm-forming ability was studied. Eighteen bacterial strains were isolated from a microbial film collected from the roof of an old house located in Sfax, Tunisia. The purity of these microorganisms was confirmed by microscopic observation after repeated streaking on a Tryptic Soy agar medium. Biofilm formation was estimated using preliminary tests including a motility test, microbial adhesion to solvents (MATS), and the Congo Red Agar method (CRA). Since these tests showed no significant result, microplate tests, such as crystal violet and resazurin assays, were used. The results obtained showed that strain S61 was able to form a biofilm within 24 h (OD₅₇₀ = 4.87). The viability of the S61 biofilm with resazurin assessed with fluorescence measurement was about 1.5 × 10³. The S61 strain was identified as Staphylococcus epidermidis. In the biofilm studied here, it was the most biofilm-forming bacterium and will be used as a bacterial model for studying anti-biofilm activity.