RpoS controls the expression and the transport of the AlgE1-7 epimerases in Azotobacter vinelandii.

Azotobacter vinelandii produces differentiated cells, called cysts, surrounded by two alginate layers, which are necessary for their desiccation resistance. This alginate contains variable proportions of guluronate residues, resulting from the activity of seven extracytoplasmic epimerases, AlgE1-7. These enzymes are exported by a system secretion encoded by the eexDEF operon; mutants lacking the AlgE1-7 epimerases, the EexDEF or the RpoS sigma factor produce alginate, but are unable to form desiccation resistant cysts. Herein, we found that RpoS was required for full transcription of the algE1-7 and eexDEF genes. We found that the AlgE1-7 protein levels were diminished in the rpoS mutant strain. In addition, the alginate produced in the absence of RpoS was more viscous in the presence of proteases, a phenotype similar to that of the eexD mutant. Primer extension analysis located two promoters for the eexDEF operon, one of them was RpoS-dependent. Thus, during encysting conditions, RpoS coordinates the expression of both the AlgE1-7 epimerases and the EexDEF protein complex responsible for their transport.

Hexuronyl C5-epimerases in alginate and glycosaminoglycan biosynthesis.

The sugar residues in most polysaccharides are incorporated as their corresponding monomers during polymerization. Here we summarize the three known exceptions to this rule, involving the biosynthesis of alginate, and the glycosaminoglycans, heparin/heparan sulfate and dermatan sulfate. Alginate is synthesized by brown seaweeds and certain bacteria, while glycosaminoglycans are produced by most animal species. In all cases one of the incorporated sugar monomers are being C5-epimerized at the polymer level, from D-mannuronic acid to L-guluronic acid in alginate, and from D-glucuronic acid to L-iduronic acid in glycosaminoglycans. Alginate epimerization modulates the mechanical properties of seaweed tissues, whereas in bacteria it seems to serve a wide range of purposes. The conformational flexibility of iduronic acid units in glycosaminoglycans promotes apposition to, and thus functional interactions with a variety of proteins at cell surfaces and in the extracellular matrix. In the bacterium Azotobacter vinelandii the alginates are being epimerized at the cell surface or in the extracellular environment by a family of evolutionary strongly related modular type and Ca(2+)-dependent epimerases (AlgE1-7). Each of these enzymes introduces a specific distribution pattern of guluronic acid residues along the polymer chains, explaining the wide structural variability observed in alginates isolated from nature. Glycosaminoglycans are synthesized in the Golgi system, through a series of reactions that include the C5-epimerization reaction along with extensive sulfation of the polymers. The single, Ca(2+)-independent, epimerase in heparin/heparan sulfate biosynthesis and the Ca(2+)-dependent dermatan sulfate epimerase(s) also generate variable epimerization patterns, depending on other polymer-modification reactions. The alginate and heparin epimerases appear unrelated at the amino acid sequence level, and have probably evolved through independent evolutionary pathways; however, hydrophobic cluster analysis indicates limited similarity. Seaweed alginates are widely used in industry, while heparin is well established in the clinic as an anticoagulant.

The catalytic activities of the bifunctional Azotobacter vinelandii mannuronan C-5-epimerase and alginate lyase AlgE7 probably originate from the same active site in the enzyme.

The Azotobacter vinelandii genome encodes a family of seven secreted Ca(2+)-dependent epimerases (AlgE1--7) catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. AlgE1--7 are composed of two types of protein modules, A and R, and the A-modules have previously been found to be sufficient for epimerization. AlgE7 is both an epimerase and an alginase, and here we show that the lyase activity is Ca(2+)-dependent and also responds similarly to the epimerases in the presence of other divalent cations. The AlgE7 lyase degraded M-rich alginates and a relatively G-rich alginate from the brown algae Macrocystis pyrifera most effectively, producing oligomers of 4 (mannuronan) to 7 units. The sequences cleaved were mainly G/MM and/or G/GM. Since G-moieties dominated at the reducing ends even when mannuronan was used as substrate, the AlgE7 epimerase probably stimulates the lyase pathway, indicating a complex interplay between the two activities. A truncated form of AlgE1 (AlgE1-1) was converted to a combined epimerase and lyase by replacing the 5'-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from algE7. Furthermore, substitution of an aspartic acid residue at position 152 with glycine in AlgE7A eliminated almost all of both the lyase and epimerase activities. Epimerization and lyase activity are believed to be mechanistically related, and the results reported here strongly support this hypothesis by suggesting that the same enzymatic site can catalyze both reactions.

A small derivative of the broad-host-range plasmid RK2 which can be switched from a replicating to a non-replicating state as a response to an externally added inducer.

TrfA is the only plasmid-encoded protein required for RK2 replication. We report here the construction and characterization of an RK2-based vector in which trfA is expressed from the inducible promoter Pm. The resulting construct, pJBSD1, was found to replicate in Escherichia coli DH5a (recA(-)) only in the presence of a Pm inducer. In two tested E. coli recA(+) strains pJBSD1 could replicate in the absence of inducer, but a replication inducer-dependent phenotype was obtained in these strains by introducing a mutation known to reduce the trfA expression level. The plasmid construct could be used as a conditional suicide vector system for targeted chromosomal integration via homologous recombination. This feature may potentially be used for many types of studies in microbial molecular biology.

The A modules of the Azotobacter vinelandii mannuronan-C-5-epimerase AlgE1 are sufficient for both epimerization and binding of Ca2+.

The industrially important polysaccharide alginate is composed of the two sugar monomers beta-D-mannuronic acid (M) and its epimer alpha-L-guluronic acid (G). In the bacterium Azotobacter vinelandii, the G residues originate from a polymer-level reaction catalyzed by one periplasmic and at least five secreted mannuronan C-5-epimerases. The secreted enzymes are composed of repeats of two protein modules designated A (385 amino acids) and R (153 amino acids). The modular structure of one of the epimerases, AlgE1, is A1R1R2R3A2R4. This enzyme has two catalytic sites for epimerization, each site introducing a different G distribution pattern, and in this article we report the DNA-level construction of a variety of truncated forms of the enzyme. Analyses of the properties of the corresponding proteins showed that an A module alone is sufficient for epimerization and that A1 catalyzed the formation of contiguous stretches of G residues in the polymer, while A2 introduces single G residues. These differences are predicted to strongly affect the physical and immunological properties of the reaction product. The epimerization reaction is Ca2+ dependent, and direct binding studies showed that both the A and R modules bind this cation. The R modules appeared to reduce the Ca2+ concentration needed for full activity and also stimulated the reaction rate when positioned both N and C terminally.

The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism.

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.

Mannuronan C-5-epimerases and their application for in vitro and in vivo design of new alginates useful in biotechnology.

The industrially important polysaccharide alginate is a linear copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). It is produced commercially by extraction from brown seaweeds, although some of the bacteria belonging to the genera Azotobacter and Pseudomonas also synthesize alginates. Alginates are synthesized as mannuronan, and varying amounts of the M residues in the polymer are then epimerized to G residues by mannuronan C-5-epimerases. The gel-forming, water-binding, and immunogenic properties of the polymer are dependent on the relative amount and sequence distribution of M and G residues. A family of seven calcium-dependent, secreted epimerases (AlgE1-7) from Azotobacter vinelandii have now been characterized, and in this paper the properties of all these enzymes are described. AlgE4 introduces alternating M and G residues into its substrate, while the remaining six enzymes introduce a mixture of continuous stretches of G residues and alternating sequences. Two of the enzymes, AlgE1 and AlgE3, are composed of two catalytically active domains, each introducing different G residue sequence patterns in alginate. These results indicate that the enzymes can be used for production of alginates with specialized properties.

Cloning and expression of three new Aazotobacter vinelandii genes closely related to a previously described gene family encoding mannuronan C-5-epimerases.

The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously been reported. The corresponding proteins catalyze the Ca2+-dependent polymer-level epimerization of beta-D-mannuronic acid to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6, algE7, and algY. All three genes were sequenced and expressed in Escherichia coli. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5'-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase. Southern blot analysis of genomic A. vinelandii DNA, using the 5' part of algE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here.

The Azotobacter vinelandii mannuronan C-5-epimerase AlgE1 consists of two separate catalytic domains.

The Azotobacter vinelandii enzyme AlgE1 is a member of a family of secreted mannuronan C-5-epimerases. These enzymes convert beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid residues (G) at the polymer level in the industrially important polysaccharide alginate, leading to altered physical and immunological properties of the polymer. The reaction product of AlgE1 was found to be a mixture of blocks of continuous G residues (G-blocks) and blocks containing alternating M and G residues (MG-blocks). The enzyme is dependent on Ca2+ for activity, and only Sr2+ of those tested was able to replace Ca2+. Zn2+ blocked the activity even at low concentrations. algE1 has been divided into two parts based on the modular type of structure previously reported to be a characteristic of the secreted epimerases, and each part has been expressed in Escherichia coli. These experiments showed that AlgE1 contains two catalytic domains, AlgE1-1, which introduces both G-blocks and MG-blocks, and AlgE1-2, which only introduces MG-blocks. AlgE1-1 has a much lower specific activity than both AlgE1-2 and AlgE1. However, the two halves of AlgE1 seem to cooperate in such a way that they contribute approximately equally to the overall epimerization reaction.

Biochemical properties and substrate specificities of a recombinantly produced Azotobacter vinelandii alginate lyase.

Alginate is a polysaccharide composed of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). An Azotobacter vinelandii alginate lyase gene, algL, was cloned, sequenced, and expressed in Escherichia coli. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL from Pseudomonas aeruginosa. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the Km was found to be 4.6 x 10(-4) M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.

Species-dependent phenotypes of replication-temperature-sensitive trfA mutants of plasmid RK2: a codon-neutral base substitution stimulates temperature sensitivity by leading to reduced levels of trfA expression.

TrfA is the only plasmid-encoded protein required for initiation of replication of the broad-host-range plasmid RK2. Here we describe the isolation of four trfA mutants temperature sensitive for replication in Pseudomonas aeruginosa. One of the mutations led to substitution of arginine 247 with cysteine. This mutant has been previously described to be temperature sensitive for replication, but poorly functional, in Escherichia coli. The remaining three mutants were identical, and each of them carried two mutations, one leading to substitution of arginine 163 with cysteine (mutation 163C) and the other a codon-neutral mutation changing the codon for glycine 235 from GGC to GGU (mutation 235). Neither of the two mutations caused a temperature-sensitive phenotype alone in P. aeruginosa, and the effect of the neutral mutation was caused by its ability to strongly reduce the trfA expression level. The double mutant and mutant 163C could not be stably maintained in E. coli, but mutant 235 could be established and, surprisingly, displayed a temperature-sensitive phenotype in this host. Mutation 235 strongly reduced the trfA expression level also in E. coli. The glycine 85 codon in trfA mRNA is GGU, and a change of this to GGC did not significantly affect expression. In addition, we found that wild-type trfA was expressed at much lower levels in E. coli than in P. aeruginosa, indicating that this level is a key parameter in the determination of the temperature-sensitive phenotypes in different species. The E. coli lacZ gene was translationally fused at the 3' end and internally in trfA, in both cases leading to elimination of the effect of mutation 235 on expression. We therefore propose that this mutation acts through an effect on mRNA structure or stability.

A new Azotobacter vinelandii mannuronan C-5-epimerase gene (algG) is part of an alg gene cluster physically organized in a manner similar to that in Pseudomonas aeruginosa.

Alginate is an unbranched polysaccharide composed of the two sugar residues beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). The M/G ratio and sequence distribution in alginates vary and are of both biological and commercial significance. We have previously shown that a family of highly related mannuronan C-5-epimerase genes (algE1 to -E5) controls these parameters in Azotobacter vinelandii, by catalyzing the Ca2+-dependent conversion of M to G at the polymer level. In this report, we describe the cloning and expression of a new A. vinelandii epimerase gene (here designated algG), localized 29 nucleotides downstream of the previously described gene algJ. Sequence alignments show that algG does not belong to the same class of genes as algE1 to -E5 but that it shares 66% sequence identity with a previously described mannuronan C-5-epimerase gene (also designated algG) from Pseudomonas aeruginosa. A. vinelandii algG was expressed in Escherichia coli, and the enzyme was found to catalyze epimerization in the absence of Ca2+, although the presence of the cation stimulated the activity moderately. Surprisingly, all activity was blocked by Zn2+. P. aeruginosa AlgG has been reported to contain an N-terminal export signal sequence which is cleaved off during expression in E. coli. This does not happen with A. vinelandii AlgG, which appears to be produced at least partly in an insoluble form when expressed at high levels in E. coli. DNA sequencing analyses of the regions flanking algG suggest that the gene is localized in a cluster of genes putatively involved in alginate biosynthesis, and the organization of this cluster appears to be the same as previously described for P. aeruginosa.

A family of modular type mannuronan C-5-epimerase genes controls alginate structure in Azotobacter vinelandii.

The L-guluronic acid residues in the Azotobacter vinelandii polysaccharide alginate originate from a post-polymerization reaction catalysed by the enzyme mannuronan C-5-epimerase (ME). We have previously reported the cloning and expression of an A. vinelandii gene encoding this enzyme, and we show here that the organism encodes at least four other ME genes originating from a common ancestor gene by a complex rearrangement process. The biological function of the corresponding enzymes is probably to catalyse the formation of alginates with a variety of physical properties. This model may explain the origin of the structural variability found in alginates isolated both from prokaryotic and eukaryotic organisms. The A. vinelandii enzymes may also potentially be useful for certain medical and biotechnological applications of this commercially important polysaccharide.

Cloning and expression of an Azotobacter vinelandii mannuronan C-5-epimerase gene.

An Azotobacter vinelandii mannuronan C-5-epimerase gene was cloned in Escherichia coli. This enzyme catalyzes the Ca(2+)-dependent epimerization of D-mannuronic acid residues in alginate to the corresponding epimer L-guluronic acid. The epimerase gene was identified by screening a bacteriophage EMBL3 gene library of A. vinelandii DNA with a synthetic oligonucleotide probe. The sequence of this probe was deduced after determination of the N-terminal amino acid sequence of a previously reported extracellular mannuronan C-5-epimerase from A. vinelandii. A DNA fragment hybridizing against the probe was subcloned in a plasmid vector in E. coli, and the corresponding recombinant plasmid expressed intracellular mannuronan C-5-epimerase in this host. The nucleotide sequence of the gene encoding the epimerase was determined, and the sequence data showed that the molecular mass of the deduced protein is 103 kDa. A module consisting of about 150 amino acids was repeated tandemly four times in the C-terminal part of the deduced protein. Each of the four repeats contained four to six tandemly oriented nonameric repeats. The sequences in these motifs are similar to the Ca(2+)-binding domains of functionally unrelated secreted proteins reported previously in other bacteria. The reaction product of the recombinant epimerase was analyzed by nuclear magnetic resonance spectroscopy, and the results showed that the guluronic acid residues were distributed in blocks along the polysaccharide chain. Such a nonrandom distribution pattern, which is important for the commercial use of alginate, has previously also been identified in the reaction product of the corresponding enzyme isolated from A. vinelandii.