Article Figures & Data
Figures
- Fig. 1.
Genetic organization of the cdg1,cdg2, and cdg3 operons and their flanking regions in A. xylinum, located on cosmid clones 3F3, 5C10, and 15A8, respectively. The pdeA and dgcgenes are indicated by solid and open boxes, respectively. Arrows indicate the transcriptional directions of the genes.
- Fig. 2.
Primer extension analysis of operoncdg2. Total RNA extracted from A. xylinum1306-3 was hybridized with 32P-end-labeled primer Cel-213. cDNA was synthesized with avian myeloblastosis virus reverse transcriptase and the four deoxyribonucleotide triphosphates. The length of the extension product (1) was measured against the terminal products produced in DNA sequencing reactions by using the same primer. G, A, T, and C indicate the individual sequencing reactions.
- Fig. 3.
Multiple alignment of the deduced amino acid sequences of the pdeA and dgc genes of A. xylinum showing the conserved GGDEF (regions I to IV) and EAL (regions V to VIII) domains. Residues identical in all six sequences are shaded in black and indicated in the consensus line; residues identical in five of six sequences are shaded in gray. In the region shown, the DGC and PDEA isoenzyme sets have 23 to 29% identity, compared to 77 to 89% identity among the PDEA sequences and 47% identity among the DGC sequences. Putative Q-linker regions are delimited by asterisks.
- Fig. 4.
Alignment of bacterial predicted protein sequences showing conservation of GGDEF and EAL domains. Residues with identity or conservative substitution in 100 and ≥80% of the sequences are shaded in black and gray, respectively. Identities displayed between each sequence and the A. xylinum (Ax) PDEA1 and DGC1 sequences, respectively, are shown in parentheses below. The amino acid sequences shown are from E. coli (Ec) YCIR, GenBank accession no. D90766 (50 and 28%); Bacillus subtilis (Bs) ykoW, GenBank Z99110 (50 and 26%); Synechocystis strain PCC6803 (Sy) slr0359, GenBank D63999 (49 and 30%);Rhizobium sp. strain NGR234 (Rh) Y4LL, GenBank AE000083 (48 and 32%); Mycobacterium tuberculosis (Mt) Y07I, GenBankZ75555 (31 and 32%).
- Fig. 5.
Multiple alignment of the N-terminal regions of the DGC1 and PDEA1 sequences showing conservation with domains on other proteins. Residues with identity or conservative substitution in 100 and ≥75% of the sequences are shaded in black and gray, respectively. (a) The amino acid sequences shown are from A. xylinum(Ax) DGC1, Synechocystis strain PCC6803 (Sy) slr1759 (sensory transduction histidine kinase, GenBank accession no. D90903 ),Klebsiella pneumoniae (Kp) NifL (GenBank X13303 ), E. coli (Ec) Aer (GenBank U28379 ), and Arabidopsis thaliana (At) NPH1 (GenBank AF030864 ). (b) Amino acid sequences shown are A. xylinum (Ax) PDEA1, E. coli(Ec) FixL (GenBank D90790 ), Rhizobium meliloti (Rm) FixL (GenBank Z70305 ), and Bradyrhizobium japonicum (Bj) FixL (GenBank P23222 ). An asterisk indicates a His residue conserved among FixL and PDEA proteins.
- Fig. 6.
Proposed domain structure of the PDEA and DGC isoenzymes of A. xylinum. The GGDEF and EAL domains form a motif conserved among all six isoenzymes. N-terminal regions putatively function in oxygen sensing (OS). Predicted sites of GTP binding may indicate catalytic sites of DGC proteins. TM, putative transmembrane helices; aa, amino acids.
Tables
- Table 1.
PDEA and DGC activities and in vivo cellulose production in A. xylinumrecombinant strainsa
Strain Site of mutation PDEA activity (%) DGC activity (%) Cellulose production (%) 1306-21 (WT) None 100 100 100 Dis1 pdeA1 20 20 85 Dis4 dgc1 98 21 80 ABT8 Upstream ofcdg1a 23 22 78 ABT9 pdeA2 85 85 98 TRT150 dgc2 98 86 95 ABT2 Partial deletion (pdeA2+dgc2) 89 91 99 ABT21 dgc1 dgc2 105 4 36 ABT11 pdeA1 pdeA2 5 5 68 ABT1 pdeA1 pdeA2 dgc2 5 4 70 ↵a Recombinant strains of A. xylinum disrupted in cdg1 and/or cdg2 were derived, and whole cells were assayed for cellulose production, as described in Material and Methods. Soluble and membrane fractions were prepared from logarithmic-phase cultures and assayed for PDEA and DGC activities.
