**2. Overview of the genetics and regulation of bacterial alginate biosynthesis**

The genetic organization of alginate-producing bacteria has been extensively analyzed and the regulation of the biosynthetic process is relatively well known, from the biosynthesis of precursor molecules, their polymerization into poly-D-mannuronic acid, chemical modifications/epimerization in the periplasmic space, and secretion to the extracellular environment. All of these steps have been previously identified and characterized in many reviews [42, 69, 70] and the biosynthesis of alginate has been shown to be extremely similar for both *Pseudomonas aeruginosa* and *Azotobacter vinelandii*, resulting from a complex regulatory network of proteins [41, 51, 59, 65]. In *P. aeruginosa*, a 12-gene operon is strictly controlled by *algD* transcription, the first gene of the alginate biosynthetic operon [53, 71], and two additional internal promoters upstream of *algG* and *algI* [72] (**Figure 1**). Three transcription promoter regions have been described for the *algD* operon, namely *algDp1* (δD promoter, *algDp2* (AlgU-δ<sup>E</sup> promoter), and *algDp3* [65]. Genes *algD* and *algA*, located downstream on the operon, are involved in the biosynthesis of the precursor guanosine diphosphate D-mannuronic acid (GDP-ManA) together with *algC*, which is found elsewhere in the bacterial chromosome, has its own promoter regions [73], and encodes for a phosphomannomutase [74]. The gene *algG* encodes for a C5-mannuronate epimerase, which converts D-ManA units into L-GulA in the periplasm [75]. Further structural modification of the nascent alginate polymer is promoted by the products of genes *algI*, *algJ*, and *algF*, all of which are involved in the O-acetylation of ManA units [76, 77]. Periplasmic proteins encoded by genes *algX*

#### **Figure 1.**

*Genetic organization of alginate biosynthesis/regulation related genes for Pseudomonas aeruginosa (blue) and Azotobacter vinelandii (green). Gene sizes are not to scale. Numbers on top of each gene indicate bp as available from https://www.ncbi.nlm.nih.gov/ for the genome of P. aeruginosa PAO1 (Ref. NC\_002516.2) and A. vinelandii DJ (Ref. NC\_012560.1). The direction of transcription and bp for alyB of A. vinelandii is still undetermined.*

and *algK*, also present as part of the operon, are supposedly involved in the guiding of alginate through the outer membrane porin (formed by *algE*) and protection of alginate against degradation of an alginate lyase encoded by *algL* [77, 78]. The polymerization process to form poly-D-ManA is promoted by two transmembrane proteins (localized on the cytoplasmic membrane) encoded by genes *alg8* and *alg44* [79, 80].

The alginate biosynthetic genes on *Azotobacter vinelandii* are also clustered and share high similarity with their previously described *P. aeruginosa* counterparts. However, despite their essentially identical functions, Pseudomonads *algE* and *algJ* genes have been designated, respectively, as *algJ* and *algV* in *A. vinelandii* (**Figure 1**). Also, *A. vinelandii* alginate gene cluster has been shown to possess two promoter regions upstream of *algD*, one of which is an AlgU (δ<sup>E</sup> )-dependent promoter, as in *P. aeruginosa*, and another RpoS (δ<sup>s</sup> )-dependent [64, 65, 81]. Additionally, other three internal promoters are found on the *A. vinelandii* operon upstream of *alg8* (δ<sup>s</sup> -dependent), *algG* (δ 70 dependent), and *algA* (regulation unknown) [64, 65]. The levels of *algA* transcription exclusively from its upstream promoter, however, are not sufficient to sustain alginate production [82]. Only in *P. aeruginosa*, the transcription of the *algA* and *algL* genes has been demonstrated to be controlled by AlgU [51].

Exclusively in *Azotobacter* spp., seven genes encoding C5-ManA epimerases have been identified, namely, *algE1-algE7* [58, 83, 84]. The translational products of these (AlgE1-AlgE7) are exported to the cell surface and released into the extracellular environment. AlgE1–7 catalyze the Ca2+-dependent epimerization of D-ManA units into L-GulA units in the alginate polymer after its secretion through the outer membrane by the AlgJ porin [51, 83]. These extracellular epimerases are structurally unrelated to AlgG, a Ca2+-independent C5-mannuronate epimerase found in the periplasm, and all seven *algE1–7* genes have been reported to be regulated by the sigma factor RpoS [85]. AlgE7 has been reported to also have lyase activity, which leads to depolymerization of alginate via β-elimination at the 4-O-glycosidic bonds [86]. In addition, four other alginate lyase genes (*alyA1–3* and *alyB*) different from *algL*, which is part of the biosynthetic operon, have been described for *A. vinelandii* [42].

#### *Bacterial Alginate Biosynthesis and Metabolism DOI: http://dx.doi.org/10.5772/intechopen.109295*

AlgU, previously called AlgT, δ<sup>E</sup> , or δ22 [87], is homologous to the alternative sigma factor of the stress response regulator RpoE from *Escherichia coli* [88] and plays a key role as the main regulator of bacterial alginate biosynthesis. AlgU ist is encoded together with the *mucABCD* operon, which contains four genes (*mucA*, *mucB*, *mucC,* and *mucD*). In both, *A. vinelandii* and *Pseudomonas* spp., the *muc* genes are located downstream of the *algU* gene, forming the *algUmucABCD* regulatory gene cluster [64, 65, 89]. Transcription of the biosynthetic *algD* operon is activated by the presence of AlgU, which in turn requires the activation of two other promoters located upstream of *algC* (*algCp1*) and an AlgU (δ<sup>E</sup> )-dependent promoter (*algDp2*) [65, 90]. In *A. vinelandii*, however, AlgU does not participate in the regulation of *algL* and *algA* genes as it has been reported for *P. aeruginosa* [51, 91]. Together, *algU* and the *mucABCD* genes form the principal regulatory switch that controls the conversion between mucoid and non-mucoid phenotypes of *P. aeruginosa* [64, 65, 89], and in other Pseudomonads, such as *P. fluorescens* and *P. syringae*, the *mucC* gene is absent and the transcription *mucD* is not dependent on AlgU [92, 93].

The regulatory proteins encoded by the *mucABCD* operon are found either embedded in the inner cell membrane or soluble in the periplasmic space. MucA is a transmembrane protein that has anti-δ<sup>E</sup> activity and acts as a negative regulator of AlgU and is required for maintaining cell viability in *P. aeruginosa* [94]. MucB has a hydrophobic cavity that interacts with MucA forming a stable complex (MucA-MucB) and serving as a fine-tune controlling mechanism that protects MucA from cleavage of its periplasmic domains by proteases [95, 96]. The role of MucC still remains to be fully determined, however, it has been hypothesized that this is an inner membrane protein that might act synergistically with MucA and MucB to negatively regulate AlgU in *A. vinelandii*, but not in *P. aeruginosa* [89, 97]. On the other hand, the role of MucD has been thoroughly determined as being a serine protease/chaperone-like protein with an important regulatory function that acts on the alginate-biosynthetic complex through AlgX [10], and also plays a central role in AlgU activation [89] functioning as a negative regulator localized in the periplasm (**Figure 2**).

Another set of genes and their respective translational products are part of a two-component signal transduction system and directly participate in the regulatory control network of alginate biosynthesis [89]. Four out of five chromosomal

#### **Figure 2.**

*Schematic representation for the genetic regulation of bacterial alginate biosynthesis. Transcription regulation (full lines) or protein-protein modulation (dashed lines) are indicated in blue (positive regulation/modulation) or red (negative modulation).*

regulatory genes (*algB*, *algP*, *algQ*, *algR*, and *algZ/amrZ*) found in Pseudomonads and *A. vinelandii* transcribe in the opposite direction from the previously described *algD*and *algUmucABCD*-operons, except for *algB*, which has the same transcriptional orientation of the biosynthetic cluster [82]. The promoter regions of *algR* and *algB* have significant homology to other promoters recognized by δ<sup>E</sup> , and these genes are positively regulated by AlgU-(δ<sup>E</sup> ) [89]. Their translational products, AlgR and AlgB, act directly on different sites of the *algD-*operon promoter in *P. aeruginosa* [98, 99]. In contrast, AlgB and AlgR are not required for activation of *algD* transcription in *A. vinelandii* [82], and mutations in *algB* did not affect alginate production for this bacterium, as recently reported [100]. The kinase AlgQ transcribed from *algQ* undergoes autophosphorylation using ATP or GTP, and transfers a phosphate group to *algR* [82]. Since AlgR binds to specific sequences of the *algD*-promoter region, it up-regulates the transcription of the biosynthesis operon in *P. aeruginosa*, together with AlgU. Although *algC* transcription is independent of other alginate genes, it has been demonstrated that *algC* is up-regulated by the presence of functional *algR* [73]. A ribbon-helix-helix DNA binding protein, *AlgZ* (also called *AmrZ* – *alginate and motility regulator*) is the product of gene *algZ* and is a strictly δ22-dependent kinase that binds to sequences localized upstream of the *algD* promoter, activating *algD* transcription [82]. It has been reported that *algZ*<sup>−</sup> mutants of *A. vinelandii* are unable to produce alginate, thus, it is proposed that AlgZ is required for the transcription of the biosynthetic *algD* operon [100], however, the molecular mechanism of the *algD* regulation by AlgZ is yet to be elucidated. AlgP was determined to be a histone-like protein that binds to the *algD* promoter and aids DNA looping to positively regulate this gene and activate translation [89].

Other regulatory systems are also present during alginate biosynthesis, including post-translational and post-transcriptional regulations [99]. A two-component system (GacS/GacA) acts as a global regulator affecting the transcription of *algD* and indirectly controlling alginate biosynthesis in *A. vinelandii* through a signaling cascade involving the post-translational system RsmA/RsmB [65]. It has been described that GacS controls the expression of *algD* from its three promoters and that *gacS*<sup>−</sup> and *gacA*<sup>−</sup> mutants of *A. vinelandii* have significantly lower levels of *algD* transcripts [51, 81]. Despite being a well-conserved regulatory system among Gram-negative bacteria, the GacS-RsmA pathway has not been reported to regulate alginate biosynthesis in *P. aeruginosa* [64, 99, 101]. It has been recently announced that the secondary messenger bis-(3′-5′) cyclic dimeric-GMP (c-di-GMP) directly regulates the transcription of the alginate operon in *P. aeruginosa* [59]. c-di-GMP also acts as a posttranslational regulator binding to PilZ domains on proteins such as the one located at the C-terminal end of Alg44 [102, 103]. The binding of c-di-GMP to the Alg44-PilZ domain is essential to activate the alginate polymerase complex Alg8-Alg44 [104].

Although the genetic organization and regulation of alginate biosynthesis seem to be similar in *Pseudomonas* spp. and *Azotobacter vinelandii*, these two genera of bacteria have strikingly different ecologies and are present in very specific habitats, which, not surprisingly, reflect the different roles alginate plays in these microorganisms. As shown above, some marked differences between these bacteria regarding alginate biosynthesis-genes distribution and regulation are well reported in the scientific literature [51, 65, 82, 99]. However, there is still much to be unveiled on the genetics and enzymatic regulatory complex for these bacteria, especially for *A. vinelandii*, which is considered a generally recognized as safe (GRAS) microorganism, with much potential for the biotechnological industry. Therefore, a broad understanding of all the biochemical and regulatory aspects of alginate biosynthesis, combined with the testing of culture conditions and molecular biology tools is paramount for the development of optimal growth methods and strains able to produce this exopolysaccharide *in vivo* or with post-production *in vitro* modifications to achieve defined G/M composition, molecular weight and ideal tailor-made physicochemical properties [46, 105–108].
