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.

Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway.

BACKGROUND: The polyene macrolide antibiotic nystatin produced by Streptomyces noursei ATCC 11455 is an important antifungal agent. The nystatin molecule contains a polyketide moiety represented by a 38-membered macrolactone ring to which the deoxysugar mycosamine is attached. Molecular cloning and characterization of the genes governing the nystatin biosynthesis is of considerable interest because this information can be used for the generation of new antifungal antibiotics. RESULTS: A DNA region of 123,580 base pairs from the S. noursei ATCC 11455 genome was isolated, sequenced and shown by gene disruption to be involved in nystatin biosynthesis. Analysis of the DNA sequence resulted in identification of six genes encoding a modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport and regulatory proteins. One of the PKS-encoding genes, nysC, was found to encode the largest (11,096 amino acids long) modular PKS described to date. Analysis of the deduced gene products allowed us to propose a model for the nystatin biosynthetic pathway in S. noursei. CONCLUSIONS: A complete set of genes responsible for the biosynthesis of the antifungal polyene antibiotic nystatin in S. noursei ATCC 11455 has been cloned and analyzed. This represents the first example of the complete DNA sequence analysis of a polyene antibiotic biosynthetic gene cluster. Manipulation of the genes identified within the cluster may potentially lead to the generation of novel polyketides and yield improvements in the production strains.

The complete nucleotide sequence of the growth-hormone gene from Atlantic salmon (Salmo salar).

We report here the complete genomic nucleotide sequence for the Atlantic salmon growth-hormone gene (asGH), including 600 bp of 5' flanking sequences. The primary transcription (3651 nt) is significantly longer than that of the mammalian genes, mainly because of larger intron sizes, but also because the asGH gene contains an additional intron (intron 5). The coding regions of the asGH gene have been compared to the corresponding regions from rainbow trout (cDNA and genomic), coho salmon (cDNA) and chum salmon (cDNA). With the exception of the rainbow trout cDNA sequence, all results were in agreement with current classification of the four species. The results of a similar comparison with the mRNA leader and trailer regions were also consistent with current classification. Sequences upstream from the transcription start point have been compared to the corresponding regions from rainbow trout and mammalian GH gene (maGH) upstream sequences. The results showed that the upstream sequences in the two fish species were very similar, while short stretches similar to conserved upstream sequences in the maGH genes were also found. Some of these conserved sequences are known to be involved in the specificity of expression of the mammalian genes.

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.

Molecular cloning and analysis of a pleiotropic regulatory gene locus from the nystatin producer Streptomyces noursei ATCC11455.

A regulatory gene locus from Streptomyces noursei ATCC14455, the producer of the antifungal antibiotic nystatin, was cloned in Streptomyces lividans based on its ability to activate actinorhodin (Act) production in this host. Deletion and DNA sequencing analyses showed that a small gene, designated ssmA, located downstream of an afsR homologue (a known pleiotropic regulator) was responsible for the Act overproduction in S. lividans. Database searches for the ssmA gene product revealed its limited similarity to the AfsR2 regulatory protein from S. lividans and CREA catabolite repressor from Aspergillus nidulans. To study the effect of ssmA on nystatin production, this gene was either deleted from S. noursei genome, or placed under control of PermE* promoter and introduced in S. noursei. The properties of the corresponding strains indicate that ssmA is involved in regulation of growth and antibiotic production only in the media with certain carbon sources.

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.

New broad-host-range promoter probe vectors based on the plasmid RK2 replicon.

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.

Ionic and acid gel formation of epimerised alginates; the effect of AlgE4.

AlgE4 is a mannuronan C5 epimerase converting homopolymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 resulted in different amounts of alternating sequences. Both ionically cross-linked alginate gels as well as alginic acid gels were prepared from the epimerised alginates. Gelling kinetics and gel equilibrium properties were recorded and compared to results obtained with the original non-epimerised alginates. An observed reduced elasticity of the alginic acid gels following epimerisation by AlgE4 seems to be explained by the generally increased acid solubility of the alternating sequences. Ionically (Ca(2+)) cross-linked gels made from epimerised alginates expressed a higher degree of syneresis compared to the native samples. An increase in the modulus of elasticity was observed in calcium saturated (diffusion set) gels whereas calcium limited, internally set alginate gels showed no change in elasticity. An increase in the sol-gel transitional rate of gels made from epimerised alginates was also observed. These results suggest an increased possibility of creating new junction zones in the epimerised alginate gel due to the increased mobility in the alginate chain segments caused by the less extended alternating sequences.

The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli.

Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-alpha2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-alpha2b expression remained poor even when fused to a signal sequence, and an alternative IFN-alpha2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

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.

Bacterial alginates: biosynthesis and applications.

Alginate is a copolymer of beta-D-mannuronic acid and alpha-L-guluronic acid (GulA), linked together by 1-4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from the genus Pseudomonas and belonging to the RNA superfamily I, are also capable of producing copious amounts of this polymer as an exopolysaccharide. The molecular genetics, regulation and biochemistry of alginate biosynthesis have been particularly well characterized in the opportunistic human pathogen Pseudomonas aeruginosa, although the biochemistry of the polymerization process is still poorly understood. In the last 3 years major aspects of the molecular genetics of alginate biosynthesis in Azotobacter vinelandii have also been reported. In both organisms the immediate precursor of polymerization is GDP-mannuronic acid, and the sugar residues in this compound are polymerized into mannuronan. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 and by epimerization of some of the residues, leading to a variable content of acetyl groups and GulA residues. In contrast, seaweed alginates are not acetylated. The nature of the epimerization steps are more complex in A. vinelandii than in P. aeruginosa, while other aspects of the biochemistry and genetics of alginate biosynthesis appear to be similar. The GulA residue content and distribution strongly affect the physicochemical properties of alginates, and the epimerization process is therefore of great interest from an applied point of view. This article presents a survey of our current knowledge of the molecular genetics and biochemistry of bacterial alginate biosynthesis, as well as of the biotechnological potential of such polymers.

Nucleotide sequence and expression analysis of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene.

The nucleotide sequence of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene was determined; this is the first procaryotic uridine diphosphoglucose pyrophosphorylase gene sequence reported. The sequence data indicated that the gene product consists of 284 amino acids. This finding was consistent with the results obtained by expression analysis in vivo and in vitro in Escherichia coli.

An experimental study on carbon flow in Escherichia coli as a function of kinetic properties and expression levels of the enzyme phosphoglucomutase.

Mutants of Escherichia coli deficient in phosphoglucomutase accumulate amylose when the cells are grown on maltose or galactose as carbon source. In the presence of physiological levels of phosphoglucomutase, most of the sugar is catabolized, leading to strongly reduced levels of amylose accumulation. By varying the expression level of heterologous phosphoglucomutase, we show that the minimum level needed to block amylose accumulation corresponds to a phosphoglucomutase activity of 150-600 nmole substrate transformed per min per mg of total soluble protein. Mutant phosphoglucomutases with strongly reduced Vmax values and increased Km values for the substrate glucose-1-phosphate or the co-substrate glucose-1,6-diphosphate, could also reduce amylose accumulation, but much higher enzyme expression levels were required.

In vitro transcription in E. coli crude lysates prepared on cellophane discs.

An in vitro RNA-synthesizing system consisting of gently lysed E. coli cells on cellophane discs is described. The system has been optimalized with respect to total RNA synthesis. Under certain standard conditions DNA dependent RNA polymerase (EC 2.7.7.6) is responsible for the majority of the RNA synthesis. The extensive rifampicin sensitivity of the synthesis indicates that most of the transcripts are initiated in vitro. The RNA synthesizing system described here has been developed with the aim of studying phage transcription in vitro. We show here that lysates of a P4 infected P2 lysogen support initiation and propagation of transcription from the P2 prophage.

The host range of RK2 minimal replicon copy-up mutants is limited by species-specific differences in the maximum tolerable copy number.

The minimal replicon of the broad-host-range plasmid RK2 consists of a gene, trfA (trans-acting replication), encoding a protein required for initiation of plasmid replication. The TrfA protein binds to iterons in the cis-acting origin of vegetative replication (oriV), but the exact mechanism by which TrfA-mediated replication initiation takes place is not known. We report here the isolation and characterization of five mini RK2 trfA mutant plasmids with an elevated plasmid copy number, four in Pseudomonas aeruginosa and one in Azotobacter vinelandii. The mutations are localized between or downstream of previously reported Escherichia coli copy-up mutations in trfA, and one of the mutations has been described earlier as an independent copy-up isolate in E. coli. The five mutant plasmids were all moderately copy up in both E. coli and their host of origin, in spite of the use of isolation procedures which were expected to select efficiently in favor of plasmid mutants specifying high copy numbers. In contrast, previously described high copy-up mutants isolated in E. coli could not be established in P. aeruginosa and A. vinelandii. These high copy-up mutants were shown to induce cell killing in E. coli under conditions where the plasmid copy number was increased as a physiological response to reduced growth rate. We propose that the reason for this killing effect is that the copy number under these conditions exceeds an upper tolerance level specific for E. coli. By assuming that the corresponding tolerance level is lower in P. aeruginosa and A. vinelandii than in E. coli, and that the mechanism of copy number regulation is similar, the model can explain the phenotypes of all tested copy up mutants in these two hosts. Analogous studies were also performed in Salmonella typhimurium and Acetobacter xylinum. The data obtained in these studies indicate that the above model is probably generally true for gram-negative bacteria, and the results also indicate that the maximum tolerable copy number is surprisingly low in some hosts.

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.

Isolation and properties of temperature-sensitive mutants of the trfA gene of the broad host range plasmid RK2.

Two small plasmid RK2 derivatives, pSV6 and pSV16, were constructed and used for the isolation and characterization of trfA mutants temperature-sensitive (ts) for replication in Escherichia coli. Four of the mutants were examined for their ability to initiate replication from the RK2 replication origin in E. coli when present in cis with respect to the origin and in trans when present on a multicopy pBR322 replicon. Each of the mutant trfA genes exhibited temperature-sensitivity in supporting replication from the RK2 origin when present in cis, and the lowest nonpermissive temperature varied depending on the mutant. When the mutant trfA genes were present on the multicopy replicon (in trans), three of the four mutant genes could support replication of the RK2-oriV plasmid pSV16 at all temperatures tested. However, with the exception of one of the mutants, the activity was reduced when compared to wild-type. The increased activity in trans possibly is the result of the increased cellular level of the TrfA protein when compared with the in cis situation where the mutant trfA gene is at a much lower copy-number. Two of the mutants also were tested in cis for temperature sensitivity in Pseudomonas aeruginosa. One of the mutants did not exhibit temperature sensitivity under the conditions employed. The second mutant showed some temperature sensitivity but the nonpermissive temperature pattern was different than that found in E. coli.

The plasmids of Acetobacter xylinum and their interaction with the host chromosome.

Acetobacter xylinum contains a complex system of plasmid DNA molecules. Plasmids of molecular weights or copy numbers different from the original wild-type, are found in different types of mutants. Restriction endonuclease digestion and DNA/DNA hybridization analysis, showed that the plasmids often contained partly, but not completely the same DNA sequences. Two of these plasmid classes were analysed in more detail, and could be shown to differ in size by about 5 kb. Hybridization analysis using cloned DNA fragments as probes, showed that sequences lacking in the smallest plasmid were still present in a DNA fraction co-migrating with linearized chromosomal DNA. In addition, at least part of the DNA in the smallest plasmid was present both in the plasmid and chromosomal DNA fraction. Analysis of a particular strain containing an insertion of transposon Tn1, also indicated the existence of complex interactions between plasmids and chromosomal DNA. Together with experiments on conjugative transfer and curing of the plasmids, the results indicate that at least part of the genetic system of A. xylinum is unusual when compared to that of other genetically characterized bacteria.

The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations.

The minimal replicon of the broad-host-range plasmid RK2 consists of the origin of vegetative replication (oriV) and a gene (trfA) encoding an essential replication protein that binds to short repeats in oriV. We report here the results of a DNA sequence analysis of seven unique mutants that are temperature sensitive for replication in Escherichia coli. The mutations (designated rts) were distributed throughout 40% of the downstream part of the trfA gene. Spontaneous revertants of the rts mutants were isolated, and further analysis of four such revertants demonstrated that the new phenotypes resulted from intragenic second-site copy up (cop) mutations. Subcloning experiments showed that all tested intragenic combinations of rts and cop mutations resulted in elimination or strong reduction of the temperature sensitivity of replication. This suppression was also observed under conditions where the mutant TrfA protein was provided in trans with respect to oriV, indicating that the reduction in temperature sensitivity could not be a TrfA protein dosage effect. The phenotypes of two of the cop mutants in Pseudomonas aeruginosa were analyzed; the results demonstrated that the mutants were either not functional or poorly functional in this host. The rts mutant plasmids were also reduced in their ability to replicate in P. aeruginosa, and the intragenic cop mutations did not improve the functionality of these mutants. The significance of the results is discussed in relation to current models of the mechanism of action of the TrfA protein.

Conjugative transfer of the naturally occurring plasmids of Acetobacter xylinum by IncP-plasmid-mediated mobilization.

Broad-host-range plasmids and cloning vectors were conjugatively transferred to Acetobacter xylinum. One of the plasmids, RP4::Mu cts61, was used for the insertion of Tn1 into the 16-, 44-, and 64-kilobase-pair plasmids of A. xylinum. The Tn1-labeled plasmids could be mobilized by a helper plasmid. Many of the Tn1 insertions affected the copy number of the plasmids.