Genetics of Bacterial Alginate: Alginate Genes Distribution, Organization and Biosynthesis in Bacteria

Abstract

Bacterial alginate genes are chromosomal and fairly widespread among rRNA homology group I Pseudomonads and Azotobacter. In both genera, the genetic pathway of alginate biosynthesis is mostly similar and the identified genes are identically organized into biosynthetic, regulatory and genetic switching clusters. In spite of these similarities,still there are transcriptional and functional variations between P. aeruginosa and A. vinelandii. In P. aeruginosa all biosynthetic genes except algC transcribe in polycistronic manner under the control of algD promoter while in A. vinelandii, these are organized into many transcriptional units. Of these, algA and algC are transcribed each from two different and algD from three different promoters. Unlike P. aeruginosa, the promoters of these transcriptional units except one of algC and algD are algT-independent. Both bacterial species carry homologous algG gene for Ca²⁺-independent epimerization. But besides algG, A. vinelandii also has algE1-7 genes which encode C-5-epimerases involved in the complex steps of Ca²⁺-dependent epimerization. A hierarchy of alginate genes expression under σ²²(algT) control exists in P. aeruginosa where algT is required for transcription of the response regulators algB and algR, which in turn are necessary for expression of algD and its downstream biosynthetic genes. Although algTmucABCD genes cluster play similar regulatory roles in both P. aeruginosa and A. vinelandii but unlike, transcription of A. vinelandii, algR is independent of σ²². These differences could be due to the fact that in A. vinelandii alginate plays a role as an integrated part in desiccation-resistant cyst which is not found in P. aeruginosa.

INTRODUCTION

Bacterial alginate is a linear exopolysaccharide consisting of β-1,4-linked β-D-mannuronic acid and its C-5 epimer α-L-guluronic acid [15,36,75,50] Bacterial alginate is partially acetylated at the O-2 and/or O-3 positions of man-nuronic acid residues [43–46,127]. Alginate polymer is primarily synthesized as polymannuronate[51,86,127] and monomers distribution is variable throughout the polymer due to epimerization of some of mannuronic acid residues [124,127]. Only two bacterial genera Pseudomonas [51,75] and Azotobacter [41,106] are known to produce alginate. In P. aeruginosa alginate acts as an extracellular matrix material that allows the formation of differentiated biofilms, which restrict diffusion of clinical antibiotics and protect embedded cells against human antibacterial defense mechanisms [78,96]. Whereas alginate produced by phytopatho-genic Pseudomonads is a virulence factor providing the encapsulated bacteria with a protective environment and being responsible for the water-soaked symptoms in infected plant tissue [116,150]. Under adverse environmental conditions A. vinelandii produces alginate both as a vegetative state capsule and as an integrated part of a particular resting stage form (desiccation-resistant cyst) [103,117]. Although the major aspects of the molecular genetics of alginate biosynthesis in A. vinelandii have also been reported [112] but most of our knowledge of the genetics of alginate biosynthesis originates from extensive studies of P. aeruginosa [110,111, 147]. Mainly because of the medical relevance of this organism as an opportunistic human pathogen that causes severe and life-threatening infections in immuno compromised hosts such as patients with respiratory diseases, burns, cancers undergoing chemotherapy, and cystic fibrosis [66,112,147]. Alginate plays an important role as a virulence factor during the infectious [50]. Therefore, P. aeruginosa has been a model for genetic studies of bacterial alginate biosynthesis. The genetics of bacterial alginate synthesis was first studied by the ability to complement a series of Alg⁻ (alginate non-producer/nonmucoid) mutants from stable Alg⁺ (alginate producer/mucoid) P. aeruginosa 8830 [19]. All P. aerugi-nosa strains carry the genes that encode the regulatory and biosynthetic machinery for alginate [137] and much knowledge on the genetic regulation of alginate biosynthesis has emerged during last two decades [51],53,112,147]. The aim of the present study is to review the independent studies and progress in molecular genetics of alginate biosynthesis and regulation in bacteria.

DISTRIBUTION OF ALIGINATE GENES

Alginate (alg) genes are distributed throughout the Pseu-domonas rRNA homology group I-Azotobacter-Azomonas lineage [39,41], while only some alg genes have been retained in the Pseudomonas group V (Xanthomonas) and enteric lineages but could not produce alginate [41]. Bacterial species belonging to Pseudomonas rRNA homology group I are actually more closely related to enteric bacteria (E. coli, K. pneumoniae) than to Pseudomonas species belonging to Pseudomonas rRNA homology groups II, III, and IV [142]. Probes for three alginate structural genes (algA, algC and algD) and one regulatory gene algR(algR1) of P. aeruginosa hybridize to DNA fragments from P. syringae pv. glycinea, P. viridiflava and P. corrugate [39] and other members of rRNA homology group I [41]. Genomic DNA from representatives of groups I-IV gave very weak or no hybridization with the probes, except for algC, indicating that the ability to produce alginate is restricted to members of rRNA homology group I. This substantiates earlier physiological studies in which alginate was isolated from P. fluorescens, P. putida, P. mendocina [59] and P. syringae [38,62]. Azomonas and Azotobacter species are closely related to Pseudomonas group I [11,29]. A. vinelandii possessed sequences homologous to all the P. aeruginosa alg genes tested, while Azo-monas macrocytogenes lacked only algD [41]. It is not known whether Azomonas species produce alginate. While the alginate-specific genes (algD and algR) would be restricted to group I Pseudomonas species or other organisms known to produce alginate [31,39]. In addition, naturally occurring alginate-producing strains of plant-associated P. fluorescens were also reported [37]. But certain phytopatho-genic fluorescent Pseudomonas species are occasionally found to produce alginate both in plants and in vitro [37,38]. All the phytopathogenic Pseudomonads tested (both Alg⁺ and Alg⁻) possess genetic sequences homologous to the P. aeruginosa alginate genes. Thus, it appears that many Pseu-domonas species are capable of producing alginate but the genes involved in alginate biosynthesis are not normally expressed [41]. However, it is difficult to be precise about the number of genes that are solely associated with alginate biosynthesis as it is now clear, for example, that some of the control genes act globally and encode proteins such as algA and algC are also essential for lipopolysaccharide (LPS) biosynthesis [58]. Similarly, homology searches of algI, algJ, and algF amino acid sequences suggested that these genes encode a family of proteins involved in the esterification of surface or extracellular polysaccharides in a variety of bacteria have evolved by lateral gene transfer [46]. Since many of the P. aeruginosa alginate genes have been cloned, it is now possible to examine genomic DNA from various Pseudo-monas species and other organisms phylogenetically near P. aeruginosa for sequences homologous to the P. aeruginosa alg genes to determine the extent to which the presence of alg gene sequences parallels phylogenetic relationships.

ORGANIZATION OF ALGINATE GENES

Irrespective of sequence homology, the physical order and organization of alg genes in A. vinelandii and alginate producing Pseudomonas species have been described identical to those of P. aeruginosa [1,72,77,90,105,110]. To date, more than 25 alg genes some of these with alternative names have been identified in P. aeruginosa and these alginate genes are all located on the bacterial chromosome, with no evidence of plasmid involvement [51,60,96,112] (Table 1, Fig. 1). In P. aeruginosa, the genes that encode the biosynthesis and regulation of alginate map to five distinct chromosomal loci [1,51,57,76,96,148] (Fig. 1). Based on their function, alg genes in P. aeruginosa are organized into three clusters namely, structural/biosynthetic, regulatory and genetic switching genes (Table 1). Virtually all of the biosyn-thetic genes including algD, alg8, alg44, algK, algE(alg76), algG, algX(alg60), algL, algI, algJ, algF, algA and algC directly catalyzing the synthesis of alginate are clustered within an 18kb region located at 34-min on the P. aerugi-nosa chromosome except algC which is located at 10-min along with a cluster of alginate regulatory genes [1,1,6,51, 80,110,118] (Fig. 1. Twelve structural genes located at 34-min are organized and transcribed as an operon with transcriptional initiating at the algD promoter, the proximal gene of the alginate biosynthesis operon [16,21,45,51,76,111] (Fig. 1). Whereas algC at 10-min transcribes in same direction but under the control of its own promoter region [151,152]. Five genes control the regulation of alginate production include algZ(fimS), algR(algR1), algQ(algR2), algP (algR3) and algB [61,76,125]. All regulatory genes are located at 9-min except algB which is located at 13-min on chromosome [15,21,28,56,96] (Fig. 1). algR is located just immediately downstream of algZ [149] and 4.5kb apart from algQ. algQ and algP are linked but not immediately adjacent [25,91]. All regulatory genes have same transcriptional orientation except algB which transcribes in the direction same as biosynthetic gene cluster [111] (Fig. 1). The alginate switching genes which mediate the conversion to constitutive Alg⁺ phenotype, consisting of five genes, algT(algU), mucA(algS), mucB(algN), mucC(algM) and mucD(algY/al gW) are clustered forming part of the same transcriptional unit at 68-min on P. aeruginosa chromosome [15,51,76,83, 85,87] (Fig. 1). Schlictman and coworkers have also studied the one more alginate regulatory gene algH but its location on chromosome is still unidentified [119].

Table 1.

Alginate Genes Clusters, Size, Mapping Sites and Products in P. aeruginosa

Gene cluster	Gene	Size (bp)	Gene product	References
Structural/biosynthetic	algD	1311	GDP-mannose dehydrogenase	[23,127,135]
	alg8	1494	subunit of alginate polymerase	[79,140]
	alg44	1170	subunit of alginate polymerase	[79,140,141]
	algK	1428	subunit of protein scafold	[64,132]
	algE(alg76)	1473	porin-like OM protein	[17,107, 148]
	algG	1632	mannuronan C-5 epimerase	[15,44,128]
	algX(alg60)	1425	subunit of protein scafold	[95,112,141]
	algL	1104	alginate lyase	[10,95,117,141]
	algI	1563	acetylase	[44,46,110]
	algJ	1176	acetylase	[44,46,110]
	algF	651	acetylase	[42,45,125]
	algA	1446	PMI-GMP	[20, 50,148]
	algC	1636	PMM	[152,152]
Regulatory	algZ(fimS)	1077	RHH DNA-binding protein	[4,5,150]
	algR(algR1)	747	respnse regulator protein of TCSTS	[24,25, 68]
	algQ (algR2)	483	cognate sensor Kinase	[66,69, 114,118]
	algP(algR3)	1058	histone-like protein	[67,70,148]
	algB	1350	NtrC subclass of TCSTS	[55, 56,144]
Genotypic switching	algT(algU)	582	sigma factor α22	[51, 84,121]
	mucA(algS)	585	anti-α22 factor	[80,84,121,122]
	mucB(algN)	951	anti-α22 factor	[80,84,121,122]
	mucC(algM)	456	homologue of PhoORF4 product	[7,8]
	mucD(algW/Y)	1425	homologue of serine protease (HtrA)	[7, 51,148]

OM(outer membrane)

PMI-GMP (phosphomannose isomerase-/guanosinediphosphomannose pyrophosphorylase)

PMM(phosphomannomutase)

PhoORF4(Photobaterium sp SS9 ORF4)

Fig. (1).

Model of the regulation of alginate biosynthesis and physical map (unshaded pentagons along with circle) of alginate structural/biosynthetic (10/34-min), regulatory (9/13-min) and genetic switching (68-min), genes at chromosome in Pseudomonas aeruginosa. Heads of pentagons indicate direction of transcription and shaded shapes in side the circular chromosome are the gene products. CSK(cognate sensor Kinase), RRP(response regulatory protein), CAP(catabolite activator protein), + and − (positive and negative effect).

GENETIC REGULATION OF ALGINATE BIOSYNTHESIS

The regulation of alginate biosynthesis is complex and involves specific gene products and those that act globally [51]. The transcriptional activation of algD gene is key point in pathway regulation of alginate synthesis and is mediated by alginate switching and regulatory genes [13,23]. Alginate gene expression is transcriptionally controlled by algT(al gU)-mucA-mucB(algN)-mucC(algM)-mucD(algW/Y) clustered at 68-min on the chromosome [85,144,146] (Fig. 1). These five genes cluster comprise the main genetic switch, controlling the conversion between non-mucoid to mucoid forms of P. aeruginosa. The algT gene encodes a 22kDa alternative sigma factor (σ²²) with significant similarity to the alternative RNA polymerase sigma factor σ^E from Es-cherichia coli and positively regulates its own promoter (PalgT) as well as the promoters of algR, algB, algD, and algC [63,85,122,125] (Fig. 1). This is because, the promoter regions preceding the transcriptional initiation sites of algT, algR, algB, algD and algC all have significant homology to promoters recognized by σ^E [33,63,83,102]. Actually, σ²²(al gU) forms a complex with RNA polymerase [76,96,122] (Fig. 1). This RNAP-σ²² complex positively regulates the transcription of algD and algR because promoters of both genes have consensus sequences for complex binding at the -35/10 region which is consistent with recognition by σ²² [30,90,120]. The other genes in the algT cluster appear to regulate the expression or activity of σ²². mucA encodes an anti-σ²² factor (Table 1) with affinity for σ²² and is a negative regulator of algT transcription [83,118,120] (Fig. 1). mucB also encodes an indirect anti-σ²² (Table 1, Fig. 1) which interacts with periplasmic domain of mucA, there by altering its conformation so that it binds σ²² and target it for degradation [62,85,122,146]. mucD encodes a periplasmic protease appears to act as a negative regulator of σ²² (Fig. 1) and has homology to the HtrA(DegP) heat shock periplasmic prote-ase of E. coli [7]. Whereas the product of mucC is homologous to that of Photobacterium sp. strain SS9 which function as negative [7,8] (Fig. 1) or alternatively as positive and negative regulator [102] but like mucB, mucC also does not directly affect algU activity [8]. Inhibition of σ²² activity by muc genes results in a nonmucoid phenotype. Spontaneous mutations in mucA mucB and mucD represent the major mechanism of conversion from the nonmucoid to the mucoid phenotype [7,8,85]. However, these mutations are unstable and spontaneous reversion to the Alg⁻phenotype often occurs due to suppressor mutations in algT [33,121,122]. Further, inactivation of the muc genes results in a high-level transcription of alginate biosynthetic and regulatory genes, such as algD and algR respectively, leading to the mucoid phenotype [7,26,81,82]. algT might not be the only sigma factor that is responsible for algD transcription. Under certain conditions, transcription of algD requires RpoN(σ⁵⁴). RpoN-dependent expression of algD is only observed in a muc23 background, where mutations are found in currently unchar-acterized gene(s) that map to a chromosomal locus distinct from mucA [9]. Besides the control exerted by genetic switching genes, the regulatory genes located at 13- and 10-min are also required for high level alginate production through the activation of algD transcription (Fig. 1). The regulatory genes algR, algB, algQ, algZ and algP express some sort of proteins which act primarily through DNA binding and bending similar to the members of bacterial two-component signal transduction systems (TCSTS) [27,28,143] (Table 1). The best characterized regulatory gene algR transcribes in response to protein σ²² and encodes response regulator protein of TCSTS which functions as positive transcriptional regulator the of genes such as algD [24,64,146], algR [69,70,96], algC [151,152] (Fig. 1) and the neuraminidase gene nanA [12]. In conjunction with algU, algR up-regulates transcription of algD and the downstream structural genes [51] (Fig. 1). algQ encodes 18kDa cognate sensor Kinase (Table 1) that undergoes autophosphorylation in the presence of ATP or GTP and transfers the acquired phosphate to algR [25,71,114,115] (Fig. 1). Functional algQ is important for algR-mediated transcriptional activation of the algD promoter [67,71,152]. algR binds three regions (named RBS) within the algD promoter at 14-mer sites having the sequence CCGTTCGTCN5′ and located upstream of the algD transcriptional initiation site [69,91,94] (Fig. 1). The first two sites are located far upstream of the algD transcriptional initiation site, at nucleotide positions −465 to −452 and −389 to −376, and have been shown to be critical for high-level expression of algD [69,130,121]. algR has also been shown to bind weakly with the algD promoter region between positions −144 and +11 [93]. There are also two sites for the binding of integration host factor (IHF) protein [138,145] as well as CysB and E. coli CAP binding sites [22,31] (Fig. 1). Mutagenesis studies suggest that both IHF and CysB play a role in algD activation, albeit much reduced in comparison with algR [22,145]. Another key alginate regulatory gene called algB is essential for high-level synthesis of alginate in P. aeruginosa [56]. Like algR, algB belongs to the response regulator superfamily of TCSTS. algB encodes a protein NtrC subclass of TCSTS (Table 1) with a molecular size of 49kDa and is required for the transcriptional activation of algD [143,145] (Fig. 1). Transcription of algB requires both the product of the algT gene and the DNA binding-bending protein IHF [144] (Fig. 1). algZ gene encodes for an absolutely σ ²²-dependent, ribbon–helix–helix (RHH) DNA binding protein proposed to be the cognate sensor kinase which binds to sequences located 280bp upstream of the algD promoter and activates the algD transcription [4,5,149]. algP encodes histone-like protein which plays a role in alginate production and might contribute to DNA looping [61] (Fig. 1). An algR mutation was constructed in FRD1, and this resulted in the loss of alginate production and a dramatic decrease in algD transcription. RNA and gene fusion analysis revealed that algB is not required for algR expression, nor is algR necessary for transcription of algB. Thus, with the exception of a requirement for algT, the algB and algR pathways appear to be independent of each other [146]. Mutation in algT blocks expression of PalgD and reduces expression of the alginate regulatory genes algR and algB [144,146]. algT or a protein under algT control also binds to sequences located within the algD promoter. Expression of the alginate biosynthetic operon requires both the response regulators (algB and algR) and algT (Fig. 1). Expression of algB and algR is dependent on algT, suggesting a circuit in which algT activates algB and algR [146]. Several proteins have been shown to bind algD sequences and are required for full transcriptional activation. As all of these genes are required, but none appear to be sufficient for full algD activation, this suggests a complex three-dimensional nucleoid structure for this promoter [5]. algC gene encodes a bifunctional enzyme with both phosphomannomutase and phosphoglucomutase activity [18,148] (Table 1) that is required not only for alginate production [148] but also for rhamnolipid production [103] and lipopolysaccharide expression [18,148]. Expression of algC is independent of other alginate genes yet algC is under the positive control of the response regulator algR in a similar manner as in case of algD [47,151] (Fig. 1). Transcriptional fusion studies of the algC promoter have shown that algC expression is reduced approximately fivefold in the absence of algR [151]. algR binds three regions positioned upstream and downstream of the transcription start site [47,130,131,151] yet the orientation and positioning of the algR binding sites differ in the algC and algD promoters. The differences in orientation of the algR binding sites and of the algR binding affinities between the algC and algD promoters and their effects on the mechanism of algR regulation in vivo have not been clearly established [131]. algR specifically bound with two regions of algC upstream DNA. A fragment spanning nucleotide positions −378 to −73 showed strong specific binding, while a fragment located between positions −73 and +187 interacted relatively weakly with algR [151]. Hence, algR regulates alginate production through binding to algD and algC promoters and interacts with RNA polymerase associated with algU [47,69,92–95,151,152] and causes a looping of promoters that is required for transcriptional activation [95].

ENVIRONMENTAL ACTIVATION

An important feature of alginate production by P. aeru-ginosa is that alginate genes are normally silent [51,57,137] but environmental stimuli like starvation, desiccation, dehydration, increasing osmolarity and the presence of phos-phorylcholine activate a cascade of regulatory proteins involved in the activation of algD promoter [6,50,135,136, 152] (Fig. 1). Further the algD promoter region shows homology to a number of bacterial promoters that are induced in response to environmental stress [136]. These include the osmoregulated promoters for E. coli outer membrane porin protein OmpF and OmpC [6,67]. In addition, the algD promoter has been found to contain a σ⁵⁴ recognition sequences [70,136]. Further more, the presence of sucrose, NaCl and KCl in the medium also activate the algD transcription [136] (Fig. 1). However, there is also a report that glucose can stimulate the algD transcription and alginate production [79]. Similarly, phosphate and nitrogen limitation conditions have also been found to activate algD in similar fashion [30–32,136] (Fig. 1). Transcriptional activation of algD promoter, the first gene in operon of structural genes, is associated with conversion to Alg⁺ phenotype. In both Alg⁺ and Alg⁻ P. aeruginosa strains, the algC promoter, like the algD promoter [6], is also activated in a hyperosmotic environment [152]. This osmolarity-induced activation is found to be dependent on algR [131,151,152]. It has been reported that the mRNA levels of algA, algC and algD genes increased, coordinately, in the cells of mucoid P. aeruginosa grown under increasing dissolved oxygen tensions of up to 70% air saturation [74]. This environmentally induced transcription of the alg genes cluster and the resulting production of alginate occur only at a low level. Copious amounts of alginate are only produced in combination with inactivation (mutations) of the negative regulators of the algU activity such as mucA, mucB, mucC and mucD [7,8,81,82,85].

ALGINATE BIOSYNTHESIS

The alginate biosynthesis pathway can be divided into four different stages: (i) synthesis of precursor substrate in the cytoplasm [125], (ii) polymerization and cytoplasmic membrane transfer, (iii) periplasmic modification, and (iv) export through the outer membrane [129,139]. The precursors synthesis which occurs in the cytoplasm is well characterized and starts from the central metabolite fructose-6-phosphate, which is converted to active precursor GDP-mannuronic acid through a series of four enzymatic steps involving the proteins encoded by algD, algA and algC genes [40,44,73,74,86,108,113,125,128,147] (Fig. 2). Fructose-6-phosphate is first isomerized into mannose-6-phos- phate by phosphomannose isopmerase encoded by algA. Mannose-6-phosphate is then converted into mannose-1-phosphate by phosphomannomutase encoded by algC [108, 148] (Table 1 and 2, Fig. 2). Then GDP-mannose is formed by the action of another enzyme GDP-mannose pyrophos-phorylase encoded by algA gene [20,51,111,147] (Fig. 2). Finally, GDP-mannose is oxidized to GDP-mannuronate by GDP-mannose dehydrogenase encoded by algD [14,87,120, 128,134] (Table 1 and 2, Fig. 2). This last step is nonrevers-ible and thought to be specific for alginate synthesis [40]. Although the polymerization steps are not well characterized but structural genes alg8 and alg44 are found to encode the catalytic subunits of the alginate polymerase localized in inner membrane which have sequence similarity to β-glyco- syltranferase [80,139,140] (Table 1 and 2). The polymeriza-tion process occurs though these proteins localized in the periplasm [64,88]. The initial polymeric product of alginate biosynthesis pathway is polymannuronate and it has been proposed that polymerization and secretion across the inner-membrane are accomplished simultaneously [87]. To produce finally biologically active structure, polymannuronate chain undergoes a series of post-polymerization modification steps carried out by a number of periplasmic proteins [40,113] (Fig. 2). Alginate is excised enzymetically by algL gene product alginate lyase from biosynthetic complex, which cleave the 1–4 glycosidic linkage by β-elimination, resulting an unsaturated nonreducing terminus [48,49,96, 118] (Table 1 and 2, Fig. 2). Although algL is required for normal production of alginate, may not be essential for polymerization in alginate synthesis [10,118] but it has been hypothesized that the alginate lyase may function either as an editing protein to control the length of the alginate polymer or to provide short pieces of alginate to prime the po-lymerization reaction [87]. The three structural genes algI, algJ, and algF encode a protein with seven transmembrane domains, a type II membrane protein, and a periplasmic protein respectively which form an enzyme complex (acetylase) (Table 1 and 2) that catalyzes the partial O-acetylation of polymeric mannuronic acid residues at the O-2 and/or O-3 positions [1,42,44,45,126] occurs either associated with the bacterial inner membrane or in the periplasm after polyman-nuronate polymerization [46]. algG encodes periplasmic mannuronan C-5 epimerase (Table 1 and 2) which is required for epimerization of some of polymeric mannuronates to guluronic acid residues in the periplasm [15,43,129] (Fig. 2). But the algG mutants of P. aeruginosa were found to produce alginate containing only polymeric D-mannuronic acid residues [15,53]. algK gene encodes a 52kDa hydrophilic protein with apparent signal peptide sequence characteristic of a lipoprotein in periplasmic subcellular localization [64] while algX encodes a hydrophilic periplasmic protein with no extensive hydrophobic domains [113] (Table 1). Recent advances indicate that a periplasmic protein complex termed as protein scaffold is formed by direct interaction of C-5 epimerase with the product of algK, and algX. This protein scaffold surrounds and protects newly formed polymers from degradation by alginate lyase as they are transported within the periplasm for further modification and eventual transport out of the cell [53,64,65,113,129,147] (Fig. 2). algE gene located between algK and algG (Fig. 1) encodes an anionic selective porin-like outer membrane protein (Table 1) which forms an alginate-specific outer membrane channel through which alginate is exported across the outer membrane into medium environment [17,44,45,108,112] (Fig. 2).

Fig. (2).

Model for the biosynthesis and assembly of alginate in P. aeruginosa [147]. I.M. and O.M(inner and outer membrane respectively), G and M(guluronate and mannuronate respectively) and Ac(acetyl group).

Table 2.

A. inelandii Alginate Genes, Known or Predicted

Gene	Products and Functions	References
algD	GDP-mannose dehydrogenase	[13,51]
alg8	subunit of alginate polymerase	[51,88]
alg44	subunit of alginate polymerase	[51,88]
algK	translocation/polymerization	[89]
algJ	porin-like OM protein	[109]
algE1-7	extracellular mannuronan C-5 epimerases	[35,54,132,133]
algY	hybrid of algE4	[132]
algG	mannuronan C-5 epimerases	[110,141]
algX	acetylation	[141]
algL	alginate lyase	[76,142]
algV	acetylation	[142]
algI	acetylation	[142]
algF	acetylation	[142]
algA	PMI-GMP	[77,141]
algC	PMM	[52,77]
algR	respnse regulator protein of TCSTS	[41,99]
algT	sigma factor α22	[84,97,100]
mucA	anti-α22 factor	[84,100,101]
mucB	anti-α22 factor	[51,84,100]
mucC	homologue of PhoORF4 product	[8,84,100,101]
mucD	homologue of serine protease (HtrA)	[51,84,100]

Alginate Genetics in A. vinelandii

In A. vinelandii our understanding of the genetics of alginate synthesis has improved greatly over the last few years but the genetic regulation has remained largely unexplored. Although, in A. vinelandii the organization of identified alginate genes and their control the alginate biosynthesis are almost in a manner similar to those of the P. aeruginosa homologs [51,72,81,90,110] but some of genes in A. vinelandii still differ in their transcriptional organization, regulation and function from P. aeruginosa (Table 1 and 2). This is most probably because A. vinelandii produces alginate both as a vegetative state capsule and as an integrated part of a particular resting stage form (cyst) of this organism [118]. Alginate biosynthetic genes cluster in A. vinelandii similar to that described in P. aeruginosa [13,77,88,89,110] is organized in at least four transcriptional units PalgD [13,88,89], Palg8-alg44-algK [88,89], PalgG-X-L-V-I-F-A and PalgA [141] (2). However, transcription of algA exclusively from promoter upstream algA is not sufficient for the algA levels required for alginate production. The later three promoters are different from the algD promoter and do not depend upon algT and algD for their activation [77,84,88, 89,141]. algD is transcribed from three promoters only one of which is indirectly algT dependent [97,101]. However, algD expression from the two algT-independent promoters is sufficient to account for alginate production [97]. But algT mutants are completely abrogated in alginate production [84,97], presumably due to the σ^E dependence of other genes involved in exopolysaccharide synthesis such as algC [52]. In contrast to P. aeruginosa, A. vinelandii does not require algR for activation of algD transcription from any of its promoters. Partial complementation of P. aeruginosa algR mutants with the A. vinelandii algR gene, however, implies that A. vinelandii algR can bind the RBS sequences present upstream of the P. aeruginosa algD promoter and can interact with algU RNA polymerase. This finding is consistent with the lack in A. vinelandii of sequences homologous to P. aeruginosa RBS [13]. It was proposed that a transcriptional regulator other than algR activates transcription of algD from the algT-dependent promoter [99]. The products of the genes alg8, alg44 and algK are candidates for subunits of the alginate polymerase complex [88,89]. The products of algX, algV, algI and algF are periplasmic proteins involved in acetylation of polymeric mannuronic acid residues at the O-2 and/or O-3 positions [141] (2). A. vinelandii algJ gene encodes an outer membrane protein (2) whose derived amino acid sequence shared approximately 52% identity with product of algE from P. aeruginosa [109]. Further, the hydrophilicity profile as well as the amphipathicity of regions in the amino acid sequence showed significant similarities to that of algE. Based on these structural similarities and topological modeling, it is believed to be functionally equivalent, forming a pore involved in alginate export [109, 111]. The alginate structural gene algC transcribes from two promoters, one of which is directly dependent on the alternative sigma factor [52]. In A. vinelandii seven unusual alginate biosynthetic genes identified as algE1-7 encode a family of seven extracellular mannuronan C-5 epimerases (AlgE1-7) [35,54,132,133] (2) which catalyze the Ca²⁺-dependent polymer-level epimerization of β-D-man-nuronic acid to α-L-guluronic acid (G) in alginate [34,132].

The algE epimerases consist of varying numbers of two types of structural modules, A and R, of which the A-module (one or two in each enzyme) alone is capable of catalyzing epimerization and determining the final distribution of G-moieties in the produced alginates [132,133]. The R-modules (one to seven copies) contain four to seven Ca²⁺-binding motifs, stimulate epimerization rates and are probably involved in export of the algE proteins [2,36]. These extracel-lular Ca²⁺-dependent epimerases generate a variety of epimerization patterns, including G blocks of various lengths. Such as algE2 catalyzes the formation of G-blocks, while algE4 introduces MG-blocks in polymeric mannuronans. algE6 introduces contiguous stretches of G residues into its substrate (G blocks), while algE7 acts as both an epimerase and a lyase [132]. The epimerase activity of algE7 leads to formation of alginates with both single G residues and G blocks [54,132,133]. Along with algE6 and algE7 another alginate gene algY was also reported which have no epime-rase activity, but a hybrid gene in which the 5′-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase [132] (2). These epime-rase genes appear to be localized in three separate physical locations, in which algE5 and algY are localized separately. Moreover, algE7 is separated by approximately 5kb from the main block containing algE1-4 and algE6. All of the enzymes therefore clearly do not originate from the same transcript. An operon organization in the block of six algE genes seems also unlikely, since the length of the region (25kb) and the spacing between the genes are larger than expected for an operon structure [132]. Surprisingly, the A. vinelandii chromosome also has an algG gene which encodes a Ca²⁺-independent periplasmic mannuronan C-5-epimerase similar to the epimerase in P. aeruginosa [43,110] (2), but its epimerization pattern has not been experimentally determined, due to low in vitro activity [54]. Sequence alignments demonstrate that algG does not belong to the algE gene family but shares 66% sequence identity to a mannuronan C-5-epimerase gene algG from P. aeruginosa [43]. In A. vine-landii and P. aeruginosa algTmucABCD gene products play similar regulatory roles in alginate biosynthesis since they are functionally interchangeable [84,100] (Table 2). In A. vinelandii, however, mucB that encodes a negative regulator of anti-σ²² factor which directly binds σ²² and target it for degradation [97,100]. mucC seems to function directly as a negative regulator of algT activity [8] but the mechanism of algT activity regulation remains to be determined. Loss-of-function mutations in either mucA or mucB have been reported to convert P. aeruginosa to mucoidy, by increasing algT activity [28,82]. In contrast, A. vinelandii strains produce alginate even in the absence of mutations in the mu-cABCD operon [101]. The algR gene from A. vinelandii has a high degree of homology with P. aeruginosa algR but the transcriptional regulation of the A. vinelandii algR gene and the role of algR in alginate production differ significantly from those of its P. aeruginosa counterparts [41,99]. These differences could be due to the fact that in A. vinelandii, alginate plays a role in encystment, a function not found in P. aeruginosa [99]. Inactivation of A. vinelandii algR diminished alginate production by 50%, but did not affect algD transcription, and completely impaired the capacity to form mature cysts. This implies that algR may exert some control over other alginate biosynthetic or regulatory genes and an unidentified biosynthetic or regulatory alginate gene other than algD is under the control of algU [97]. Transcription of algR is not abrogated in the algU mutant strains; accordingly, the algR promoter does not have algU consensus sequences. This indicates the potential existence of another sigma factor involved in alginate and encystment control [99]. Whether phosphorylation of A. vinelandii algR is necessary for activation of its target cyst promoters remains to be investigated. Further studies will help to clarify the role of a response regulator such as algR in signal transduction and its interaction with algU-RNA polymerase.

CONCLUSION

Alginate production in bacteria is under a complex regulatory control of a number of genes products, environmental stimuli and other factors. The genes involved in bacterial alginate production are found and can express only in rRNA homology group I Pseudomonads and Azotobacter representatives. In these bacteria, all alginate genes are chromosomal and most of them are transcriptionally organized into many operons. Much knowledge of alginate genetics has been gained from P. aeruginosa than A. vinelandii and most of genetic aspects such as gene organization, function and transcription appear to similar but still some significant differences are there. The biosynthetic genes cluster in P. aerugi-nosa is under transcriptional control of algD except algC at 10-min whereas in A. vinelandii genes of biosynthetic cluster function as many transcriptional units. Further, A. vinelandii algC and algD have more than one functional promoters each behaves differently. Similarly in A. vinelandii epimerization steps are more complex and beside algG, seven different genes (algE1-7) products are involved than P. aerugi-nosa. In P. aeruginosa the transcriptional regulation of algR, algB, algC and algD is algT dependent whereas in A. vine-landii these genes transcribe independent of algT except one of promoters of each algC and algD. Although the most of genetic aspects of bacterial alginate have been extensively studied since last two decades and much knowledge has been gained. But still gabs are there especially the mechanism of polymerization and post polymerization modifications, is not well-understood. The ongoing research all over the world soon shall expectedly fill these unexplored gabs in near future.