Identification and characterization of an Azotobacter vinelandii type I secretion system responsible for export of the AlgE-type mannuronan C-5-epimerases

Abstract

Alginate is a linear copolymer of beta-d-mannuronic acid and its C-5-epimer, alpha-l-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (< or =2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.

FIG. 1.

Phylogenetic analysis of A regions of putative ABC proteins in the A. vinelandii OP genome. Individual A regions are indicated with gene/protein numbers as annotated in the draft genome sequence. Both A regions of experimentally verified type I protein exporters from other bacteria (▴) and A regions of putative A. vinelandii exporters are typed in black. One putative A region appears not to be involved in membrane transport (typed in gray and marked with •), and putative ABC proteins predicted to be involved in import are typed in gray. Individual A regions from proteins with two ABCs fused in tandem are indicated with α and β. A regions from ABCs fused to an MD are indicated with an asterisk. Classifications of the closest BLAST hit for each protein in TCDB (50), when blasting with the full protein sequence, are indicated in parentheses. Clusters of similar A regions are numbered (I to X), and expected substrate specificities within each cluster, according to closest hits in the TCDB, are indicated after each cluster number.

FIG. 2.

Organization of eexD, eexE, and eexF genes. (A) Orientations and sequential arrangement of eexD, eexE, and eexF. InterPro identifiers detected by InterProScan are indicated (IPR011527, ABC transporter transmembrane region; IPR003439, ABC transporter-related domain; IPR010128, type I secretion system ATPase PrtD; IPR010129, type I secretion membrane fusion protein HlyD; IPR010130, type I secretion outer membrane protein TolC). (B and C) Nucleotide sequences between genes eexD and eexE and between genes eexE and eexF, respectively. Sequences corresponding to possible start (ATG and GTG) and stop codons (TGA) are underlined, and the numbers of amino acid residues of the corresponding deduced proteins are indicated in parentheses above each start. Start sites were predicted by GeneMarkS analysis (3), signal sequence analysis (SignalP 3.0) (2), and when applicable, alignment with the closest homologues when protein-protein blasted against the nonredundant database. The corresponding start sites are indicated by γ, β, and α, respectively. All of these approaches indicated that an eexD ORF corresponds to a protein of 580 amino acids.

FIG. 3.

Western blot detection of AlgE MEs in culture supernatants at different stages of growth after replacement of Burk glucose medium with Burk BHB (0.2%). (A and B) Signals after 24 and 176 h of growth, respectively. Lane 1, molecular mass standard (Bio-Rad Precision Plus Dual Color); lane 2, A. vinelandii E wild-type strain; lane 3, AvE109; lane 4, AvE152; lane 5, AvE153. The identities of individual bands cannot be determined directly due to the abnormal mobility of the AlgE MEs (27).

FIG. 4.

¹H NMR spectra of alginates harvested from the A. vinelandii E wild-type strain and the eex mutants (AvE109, AvE152, and AvE153) grown in RA1 medium. The GG-5M, GG-5G, and MG-5G signals, which identify G block alginates, are visible in wild-type alginate and absent from all of the mutant alginates. All spectra were expanded horizontally, and the tops of the M-1M peaks are not shown in order to reduce the picture size. The monad and diad frequencies (internal residues) of each alginate are summarized at the top of the figure. F_GM, fraction of G residues with M as the nearest neighbor; F_MM, fraction of M residues with M as the nearest neighbor.

FIG. 5.

Cell morphologies of the A. vinelandii E wild-type strain and eex mutant derivatives (here exemplified by AvE109) grown in RA1 medium. (A) Cell morphologies as a function of time (light microscopy). Samples were withdrawn from each culture and stained as described by Vela and Wyss (59). The number of days of growth is indicated in the left column. The optical densities of each culture at 600 nm are indicated in parentheses in each picture. (B and C) Electron transmission micrographs of A. vinelandii E wild type and mutant AvE109, respectively, grown in RA1 medium. Ct, cell coat; Db, coat debris; Gr, intracellular granules.

FIG. 6.

Effect of addition of proteases to A. vinelandii E wild-type strain and eex mutant culture media. (A) Visualization of viscosity of wild-type and eex mutant RA1 culture samples with (+) and without (−) proteases. The cultures were grown in shake flasks, and samples were transferred to the tubes. Tube 1, A. vinelandii E wild type (−); tube 2, A. vinelandii E wild type (+); tube 3, AvE109 (−); tube 4, AvE109 (+); tube 5, AvE152 (−); tube 6, AvE152 (+); tube 7, AvE153 (−); tube 8, AvE153 (+). (B) Molecular masses of wild-type and eex mutant alginates produced in RA1 medium with and without proteases. Molecular masses above 1,000 kDa could not be accurately determined (marked with an asterisk).