The elevated expression of the norA gene is responsible for efflux-mediated resistance to quinolones in Staphylococcus aureus (E.Y.W. Ng, M. Trucksis, and D.C. Hooper, Antimicrob. Agents Chemother. 38:1345-1355, 1994). For S. aureus transformed with a plasmid containing the cloned norA gene, SA113(pTUS20) (H. Yoshida, M. Bogaki, S. Nakamura, K. Ubukata, and M. Konno, J. Bacteriol. 172:6942-6949, 1990), and an overexpressed mutant, SA-1199B (G.W. Kaatz, S.M. Seo, and C.A. Ruble, J. Infect. Dis. 163:1080-1086, 1991), the MICs of norfloxacin increased 16 and 64 times compared with its MICs for the recipient and wild-type strains, SA113 and SA-1199, respectively. MICs of CS-940, however, increased only two and eight times, even though these two fluoroquinolones are similarly hydrophilic (apparent logPs of approximately -1). No good correlation was found, among 15 developed and developing quinolones, between the increment ratio in MICs and hydrophobicity (r = 0.61). Analysis of the quantitative structure-activity relationship among 40 fluoroquinolones revealed that the MIC increment ratio was significantly correlated with the bulkiness of the C-7 substituent and bulkiness and hydrophobicity of the C-8 substituent of fluoroquinolones (r = 0.87) and not with its molecular hydrophobicity (r = 0.47). Cellular accumulation of norfloxacin in SA-1199B was significantly lower than that in SA-1199, and it was increased by addition of carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, accumulations of CS-940 in these strains were nearly identical, and they were not affected by addition of the protonophore.

Filamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that α-1,3-glucan functions in hyphal aggregation. However, Aspergillus oryzae α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in these mutants. Here, we identified an extracellular matrix polysaccharide galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae (AG-GAGΔ), we disrupted the genes required for GAG biosynthesis in an AGΔ mutant. Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG fraction to the hyphae of the AG-GAGΔ strain resulted in formation of mycelial pellets. Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, suggesting that hydrogen bond formation by this group is important for aggregation. Genome sequences suggest that α-1,3-glucan, GAG, or both are present in many filamentous fungi and thus may function in hyphal aggregation in these fungi. We also demonstrated that production of a recombinant polyesterase, CutL1, was higher in the AG-GAGΔ strain than in the wild-type and AGΔ strains. Thus, controlling hyphal aggregation factors of filamentous fungi may increase productivity in the fermentation industry.

226 words 2 Text, 5,353 words 3 3Abstract Filamentous fungi generally form aggregated hyphal pellets in liquid culture. 1 We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did 2 not form hyphal pellets and their hyphae were fully dispersed, and we suggested that 3 α-1,3-glucan functions in hyphal aggregation. Yet, Aspergillus oryzae 4 α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we 5 hypothesized that another factor responsible for forming hyphal pellets remains in these 6 mutants. Here, we identified an extracellular matrix polysaccharide 7 galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae 8 (AG-GAGΔ), we disrupted the genes required for GAG biosynthesis in an AGΔ mutant. 9 Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that 10 GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG 11 fraction to the hyphae of the AG-GAGΔ strain resulted in formation of mycelial pellets. 12 Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, 13 suggesting that hydrogen bond formation by this group is important for aggregation. 14 Genome sequences suggest that α-1,3-glucan, GAG, or both are present in many 15 filamentous fungi and thus may function in hyphal aggregation in these fungi. We also 16 demonstrated that production of a recombinant polyesterase, CutL1, was higher in the 17 AG-GAGΔ strain than in the wild-type and AGΔ strains. Thus, controlling hyphal 18 aggregation factors of filamentous fungi may increase productivity in the fermentation 19 industry. 20 21 22Importance Production using filamentous fungi is an important part of the 1 fermentation industry, but hyphal aggregation in these fungi in liquid culture limits 2 productivity compared with that of yeast or bacterial cells. We found that 3 galactosaminogalactan and α-1,3-glucan both function in hyphal aggregation in 4 Aspergillus oryzae, and that the hyphae of a double mutant deficient in both 5 polysaccharides become fully dispersed in liquid culture. We also revealed the relative 6 contribution of α-1,3-glucan and galactosaminogalactan to hyphal aggregation. 7 Recombinant protein production was higher in the double mutant than in the wild-type 8 strain. Our research provides a potential technical innovation for the fermentation 9 industry that uses filamentous fungi, as regulation of the growth characteristics of A.10 oryzae in liquid culture may increase productivity. 11 12 13