Soledad Moreno

The GacS Sensor Kinase Regulates Alginate and Poly-β-Hydroxybutyrate Production in Azotobacter vinelandii

Azotobacter vinelandii produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-beta-hydroxybutyrate (PHB). A cosmid clone (pSMU588) from an A. vinelandii gene library diminished alginate production by A. vinelandii mucoid strain ATCC 9046. The nucleotide sequence and predicted amino acid sequence of the locus responsible for the mucoidy suppression revealed 65% identity to Pseudomonas GacS, a transmembrane sensor kinase of the two-component regulators, whose cognate response regulator, GacA, is a global activator regulating several products and virulence factors. Plasmid pMC15, harboring gacS, and a strain carrying a gacS nonpolar mutation were constructed. Either pMC15 or the gacS mutation significantly reduced alginate production and transcription of algD, the gene coding for the key enzyme GDP-mannose dehydrogenase of the alginate biosynthetic pathway. We found that the gacS mutation also reduced PHB accumulation and impaired encystment. Taken together, these data indicate that in A. vinelandii the gacSA global system regulates polymer synthesis.

The global regulators GacA and sigma(S) form part of a cascade that controls alginate production in Azotobacter vinelandii.

Transcription of the Azotobacter vinelandii algD gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3. The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of algD from its three sites during the stationary phase. Here we show that algD is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase. We constructed A. vinelandii strains that carried mutations in gacA encoding the cognate response regulator of GacS and in rpoS coding for the stationary-phase sigma(S) factor. The gacA mutation impaired alginate production and transcription of algD from its three promoters. Transcription of rpoS was also abolished by the gacA mutation. The rpoS mutation impaired transcription of algD from the p1 promoter and increased it from the p2 sigma(E) promoter. The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in A. vinelandii.

Enzyme INtr, NPr and IIANtr Are Involved in Regulation of the Poly-β-Hydroxybutyrate Biosynthetic Genes in Azotobacter vinelandii

The ptsP, ptsO, and ptsN genes encode Enzyme INtr, NPr, and enzyme IIANtr (IIANtr) proteins of the nitrogen-related phosphotransferase system. These proteins participate in a phosphoryl transfer chain in several bacteria, where IIANtr appears to be the terminal phosphoryl acceptor. Inactivation of the ptsP gene in Azotobacter vinelandii was previously shown to reduce poly-β-hydroxybutyrate (PHB) production. Therefore, the question of a role of the ptsO and ptsN gene products in PHB synthesis was raised. In this work we constructed strains carrying mutations in the ptsO and ptsN genes and tested their effects on PHB accumulation. In the ptsO mutant, PHB accumulation diminished as in the ptsP mutant, while the ptsN mutant accumulated more PHB than the wild-type strain. The negative effects of the ptsP and ptsO mutations on PHB accumulation was suppressed by the ptsN mutation, and a H68A mutation in the phosphorylatable site of IIANtr, impaired PHB accumulation similar to the ptsP mutation. The ptsP and ptsO mutations negatively affected transcription of the phbBAC biosynthetic operon and of the phbR gene coding for a transcriptional activator of phbBAC, whereas the ptsN mutation increased expression of this operon. Taken together our data provide genetic evidence suggesting that the non-phosphorylated form of IIANtr is involved in negative regulation of phbR and phbBAC expression in A. vinelandii.

Genetic analysis of the transcriptional arrangement of Azotobacter vinelandii alginate biosynthetic genes: identification of two independent promoters

The study of alginate biosynthesis, the exopolysac charide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, might lead to different bio‐technological applications. Here we report the cloning of A. vinelandii algA, the gene coding for the bifunctional enzyme phosphomannose isomerase‐guano‐sine diphospho‐D‐mannose pyrophosphorylase (PMI‐GMP). This gene was selected by the complementation for xanthan gum production of Xanthomonas campestris pv. campestris xanB mutants, which lack this enzymatic activity. The complementing cosmid clones selected, besides containing algA, presented a gene coding for an alginate lyase activity (algL), and some of them also contained algD which codes for GDP‐mannose dehydrogenase. We present here the characterization of the A. vinelandii chromosomal region comprising algD and its promoter region, algA and algL, showing that, as previously reported for P. aeruginosa, A. vinelandii has a cluster of the biosynthetic alginate genes. We provide evidence for the presence of an algD‐independent promoter in this region which transcribes at least algL and algA, and which is regulated in a manner that differs from that of the algD promoter.

The Azotobacter vinelandii alg8 and alg44 genes are essential for alginate synthesis and can be transcribed from an algD-independent promoter

A 2.8-kb DNA region, located immediately downstream of algD, contains the A. vinelandii alg8 and alg44 genes, whose sequences are highly homologous to those of the corresponding Pseudomonas aeruginosa genes. These genes occur on a transcript that does not include algD, and are transcribed from a promoter different from that transcribing algD; this is the fourth promoter described within the alginate biosynthetic gene cluster. alg8 and alg44 mutants were constructed and shown to be completely impaired in alginate production. Alg8 shares 28.20% identity and 38.09% similarity to Azorhizobium caulinodans NodC, a glycosyl transferase catalyzing the formation of beta-1,4 linkages. A topological model is predicted, which supports the idea of Alg8 being the polymerase responsible for alginate synthesis.

Transcriptional organization of the Azotobacter vinelandii algGXLVIFA genes: characterization of algF mutants

Azotobacter vinelandii forms desiccation-resistant cysts which contain a high proportion of the exopolysaccharide alginate in their envelope. We have previously shown that the A. vinelandii alginate biosynthetic genes algA and algL are transcribed from a promoter located somewhere upstream of algL. In this study we sequenced the A. vinelandii algX, algL, algV, algI and algF genes located between algG and algA. We carried out primer extension analysis of the algG, algX and algL genes and detected transcription start sites upstream algG but not upstream algX or algL, implying that algG and algX form part of the previously identified algL-A operon. A promoter upstream algA was also detected; however, transcription of algA exclusively from this promoter is not sufficient for the AlgA levels required for alginate production. An algF mutant (AJ34) was constructed by insertion of the Omega-tetracycline cassette in the non-polar orientation. As expected, AJ34 produced unacetylated alginate. Viability of 35day old cysts formed by strain AJ34, but not of those formed by the wild type, was reduced, indicating that acetylation of alginate plays a role in cyst resistance to desiccation.

RsmA post-transcriptionally controls PhbR expression and polyhydroxybutyrate biosynthesis in Azotobacter vinelandii

In Azotobacter vinelandii the two-component GacS/GacA system is required for synthesis of polyhydroxybutyrate (PHB) and of the exopolysaccharide alginate. The RsmA protein was shown to interact with the alginate biosynthetic algD mRNA, acting as a translational repressor, and GacA was found to activate transcription of the rsmZ1 and rsmZ2 genes that encode small RNAs interacting with RsmA to counteract its repressor activity. The phbBAC operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR. This study shows that GacA is required for transcription of one rsmY and seven rsmZ1-rsmZ7 genes present in the A. vinelandii genome, and that inactivation of rsmA results in increased PHB production. Transcriptional and translational phbR-gusA gene fusions were used to show that the gacA mutation negatively affected the expression of the phbR gene at the translational level. We also demonstrated an in vitro interaction of RsmA with RNAs corresponding to phbB and phbR mRNA leaders, and showed that the stability of phbR and phbB mRNAs is increased in the rsmA mutant. Taken together these results indicate that in A. vinelandii, RsmA post-transcriptionally represses the expression of PhbR.

The Azotobacter vinelandii AlgE mannuronan C-5-epimerase family is essential for the in vivo control of alginate monomer composition and for functional cyst formation

The industrially widely used polysaccharide alginate is a co-polymer of beta-D-mannuronic acid and alpha-L-guluronic acid (G), and the G residues originate from a polymer-level epimerization process catalysed by mannuronan C-5-epimerases. In the genome of the alginate-producing bacterium Azotobacter vinelandii genes encoding one periplasmic (AlgG) and seven secreted such epimerases (AlgE1-7) have been identified. Here we report the generation of a strain (MS163171) in which all the algE genes were inactivated by deletion (algE1-4 and algE6-7) or interruption (algE5). Shake flask-grown MS163171 produced a polymer containing less than 2% G (algG still active), while wild-type alginates contained 25% G. Interestingly, addition of proteases to the MS163171 growth medium resulted in a strong increase in the chain lengths of the alginates produced. MS163171 was found to be unable to form functional cysts, which is a desiccation-resistant differentiated form developed by A. vinelandii under certain environmental conditions. We also generated mutants carrying interruptions in each separate algE gene, and a strain containing algE5 only. Studies of these mutants indicated that single algE gene inactivations, with the exception of algE3, did not affect the fractional G content much. However, for all strains tested the alginate composition varied somewhat as a response to the growth conditions.

Identification and Characterization of an Azotobacter vinelandii Type I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

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.

Isolation and Characterization of Azotobacter vinelandii Mutants Impaired in Alkylresorcinol Synthesis: Alkylresorcinols Are Not Essential for Cyst Desiccation Resistance

During encystment of Azotobacter vinelandii, a family of alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the mature cyst, these lipids replace the membrane phospholipids and are also components of the layers covering the cyst. In this study, A. vinelandii strains unable to synthesize ARs were isolated after mini-Tn5 mutagenesis. Cloning and nucleotide sequencing of the affected loci revealed the presence of the transposons within the arsA gene of the previously reported arsABCD gene cluster, which encodes a type I fatty acid synthase. A mutant strain (SW-A) carrying an arsA mutation allowing transcription of arsBCD was constructed and shown to be unable to produce ARs, indicating that the ArsA protein is essential for the synthesis of these phenolic lipids. Transcription of arsA was induced 200-fold in cells undergoing encystment, but only 14-fold in aged cultures of A. vinelandii, in accordance with AR synthesis and cyst formation percentages under the two conditions. Although it was previously reported that the inactivation of arsB abolishes AR synthesis and results in a failure in encystment, the arsA mutants were able to form cysts resistant to desiccation. These data indicate that ARs play a structural role in the exine layer of the cysts, but they are not essential for either cyst formation or for desiccation resistance.