Identification and characterization of non-cellulose-producing mutants of Gluconacetobacter hansenii generated by Tn5 transposon mutagenesis

Abstract

The acs operon of Gluconacetobacter 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 Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel(-)) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript "Ax" indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel(-) mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel(-) mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis.

Fig 1

Schematics of genes inserted by Tn5 transposon DNA into six Cel⁻ mutants. Dark vertical arrows indicate the sites of insertion. For acsAB, the regions encoding the truncated proteins used to raise anti-AcsA_cat and anti-AcsB antibodies and the region encoding the PilZ domain are indicated. For acsC, the regions encoding two peptides used to raise anti-AcsC antibody are indicated. Broken vertical arrows indicate the putative cleavage sites of AcsAB as it is processed to AcsA_cat, AcsA_reg, and AcsB.

Fig 2

Protein blot analysis of Acs proteins in the wild type (WT) and five Cel⁻ mutants. For each mutant, the gene disrupted is indicated in parentheses. Equal amounts (40 μg) of total proteins were used for all samples. The antibody used for each blot is shown to the left.

Fig 3

Amount of cellulose produced by each complemented Cel⁻ mutant relative to that of the wild type (WT). Each Cel⁻ mutant was transformed with pUCD2 carrying a functional copy of the gene disrupted by Tn5 transposon. The promoter of the acs operon (acsp) was used to drive the expression acsC in mutant 10. For all of the other mutants, either the entire operon (mutants 5 and I-13) or the entire gene driven by its native promoter (mutants I-7 and II-23) was introduced. After growth in 100 ml of SH medium with spectinomycin (100 μg/ml) and tetracycline (20 μg/ml) under static conditions for 7 days, equal amounts of cells, based on the OD₆₀₀, were used for determining the dry weights of cellulose present in the medium. Each bar represents the percentage of the mean of three biological replicates of a complemented mutant relative to that of the wild type. Error bars represent means ± the standard deviations (SD).

Fig 4

Protein blot analysis of Acs proteins produced in the wild type (WT) and complemented Cel⁻ mutants. The gene disrupted in each mutant is indicated in parentheses, and the antibody used for each blot is shown on the left. For each mutant, its complemented transformant is indicated by a subscript “CE”.

Fig 5

Growth curves of Cel⁻ mutants and WT (Cel⁻), the stable non-cellulose-producing clone of the wild type. The gene disrupted in each mutant is indicated in parentheses. Cells were grown under static conditions in 100 ml of SH medium with tetracycline (20 μg/ml), and the OD₆₀₀ values were taken for all of the samples at the same time on each day during a 15-day period.

Fig 6

Quantitative RT-PCR analysis of transcript levels of cmc_Ax, ccp_Ax, bgl_Ax, dgc1, and crp-fnr in mutant II-23 (A) and in the wild type carrying a functional copy of crp-fnr in pUCD2 (B). The data are normalized to 16S RNA levels, and for each gene in panels A and B the data are presented as a level relative to that of the wild type, denoted by the dashed line at 1.0. Three independent biological replicates were used for each gene and, for each biological replicate, at least three qRT-PCRs were performed. Error bars represent means ± the SD.

Fig 7

Protein blot analysis of Bgl_Ax. (A) Wild type (WT) and five Cel⁻ mutants. The gene disrupted in each mutant is indicated in parentheses. (B) Mutants II-23, II-23_CE (complemented mutant II-23), II-23_OE (WT transformed with a functional copy of crp-fnr in pUCD2), and bgl_Ax⁻ (a bgl_Ax knockout mutant). An anti-Bgl_Ax antibody was used for both blots.