초록
<P>The <I>acs</I> operon of <I>Gluconacetobacter</I> is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn<I>5</I> transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel<SUP>−</SUP>) mutants of <I>Gluconacetobacter hansenii</I> ATCC 23769. The genes disrupted were <I>acsA</I>, <I>acsC</I>, <I>ccp<SUB>Ax</SUB></I> (encoding cellulose-complementing protein [the subscript “<I>Ax</I>” indicates genes from organisms formerly classified as <I>Acetobacter xylinum</I>]), <I>dgc1</I> (encoding guanylate dicyclase), and <I>crp-fnr</I> (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the <I>acsA</I> mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the <I>ccp<SUB>Ax</SUB></I> mutant, and (iii) the level of AcsD was not affected in any of the Cel<SUP>−</SUP> mutants. Promoter analysis showed that the <I>acs</I> operon does not include <I>acsD</I>, unlike the organization of the <I>acs</I> operon of several strains of closely related <I>Gluconacetobacter xylinus</I>. Complementation experiments confirmed that the gene disrupted in each Cel<SUP>−</SUP> mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of <I>bgl<SUB>Ax</SUB></I> (encoding β-glucosidase and located immediately downstream from <I>acsD</I>) was strongly dependent on Crp/Fnr. A <I>bgl<SUB>Ax</SUB></I> knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the <I>crp-fnr</I> mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis.</P>