Nucleotide sequence of levansucrase gene (levU) of Zymomonas mobilis ZM1 (ATCC10988)

Song, Kyung-Sik; Joo, Hyun-Kyu; Rhee, Shin Hyung

doi:10.1016/0167-4781(93)90130-6

Cited by 57 publications

(42 citation statements)

References 15 publications

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“…The LsdA signal peptide has the essential features of a Secdependent signal peptide. By contrast, other gram-negative bacteria, including the close relative Gluconacetobacter xylinus, secrete levansucrases by a signal-peptide-independent pathway (14,18,23,41,42,45). Some of these enzymes have been localized and are fully active in the periplasm of the native host or a recombinant E. coli strain (14,18,19,23), and the occurrence of a type II secretion pathway responsible for their transfer across the outer membrane cannot be ruled out.…”

Section: Discussionmentioning

confidence: 99%

“…Levansucrase secretion in gram-positive bacteria involves cleavage of signal-peptidecontaining precursors in Bacillus subtilis (43), Bacillus amyloliquefaciens (46), Geobacillus stearothermophilus (24), Paenibacillus polymyxa (7), Streptococcus salivarius (34), Actinomyces naeslundii (6), Arthrobacter nicotinovorans (36), and Lactobacillus reuteri (49). However, with the exception of LsdA, the other gram-negative levansucrases characterized so far are secreted by a signal-peptide-independent pathway; this occurs in Zymomonas mobilis (41), Erwinia amylovora (14), Rahnella aquatilis (42), Pseudomonas syringae (18,23), and Gluconacetobacter xylinus (45). LsdA is synthesized as a precursor with a 30-residue N-terminal signal peptide, which is cleaved off during transport to the periplasm, where the enzyme adopts its final conformation.…”

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confidence: 99%

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A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

et al. 2004

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The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, Gluconacetobacter (formerly Acetobacter) diazotrophicus is a nonpathogenic, nitrogen-fixing endophyte of sugarcane and other predominantly sucrose-rich crops (11,20,47). This gramnegative bacterium lacks a sucrose transport system (3) and depends on the secretion of a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose (16,17). The levansucrase gene (lsdA) and an exolevanase gene (lsdB) downstream form a chromosomal operon with a high level of conservation among G. diazotrophicus isolates (17, 26).-All known levansucrases are extracellular proteins, although they follow different secretion routes. Levansucrase secretion in gram-positive bacteria involves cleavage of signal-peptidecontaining precursors in Bacillus subtilis (43), Bacillus amyloliquefaciens (46), Geobacillus stearothermophilus (24), Paenibacillus polymyxa (7), Streptococcus salivarius (34), Actinomyces naeslundii (6), Arthrobacter nicotinovorans (36), and Lactobacillus reuteri (49). However, with the exception of LsdA, the other gram-negative levansucrases characterized so far are secreted by a signal-peptide-independent pathway; this occurs in Zymomonas mobilis (41), Erwinia amylovora (14), Rahnella aquatilis (42), Pseudomonas syringae (18, 23), and Gluconacetobacter xylinus (45). LsdA is synthesized as a precursor with a 30-residue N-terminal signal peptide, which is cleaved off during transport to the periplasm, where the enzyme adopts its final conformation. Then, in a rate-limiting step, the mature LsdA is transferred across the outer membrane and released into the extracellular medium without further proteolytic cleavage (15).A wide spectrum of gram-negative bacteria, mainly plantand animal-interacting species, utilize the type II secretion pathway for extracellular release of particular proteins, such as hydrolytic enzymes and toxins. Following translocation to the periplasm by the signal-peptide-dependent Sec or TAT system (32, 52), the folded substrate protein is transported across the outer membrane by the type II secretion apparatus, consisting of a multiprotein complex of at least 12 components (reviewed in reference 38).Here we report that the endophytic bacterium G. diazotrophicus possesses a type II secretion operon required for LsdA transport across the outer membrane. The locus was identified downstream of the levansucrase-levanase (lsdA-lsdB) operon on the chromosome of the 14 strains tested, recovered from different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II secretion pathway in the Acetobacteraceae. MATERIALS AND METHODSBacterial strains, media, and plasmids....

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

et al. 2004

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“…So far, all levan-producing bacteria tested have contained a single gene coding for Lsc in their genomes (2,15,16,21,(43)(44)(45). Redundance of lsc in the genome of P. syringae could signal its ecological importance or might hint at different functions for each of the gene products.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to levansucrases from gram-positive bacteria, which differ widely in their biochemical characteristics, levansucrases from gramnegative bacteria are similar in their molecular mass and substrate-independent expression (16,22,23,43).…”

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confidence: 99%

Characterization and Mutational Analysis of Three Allelic lsc Genes Encoding Levansucrase in Pseudomonas syringae

Ullrich

2001

J Bacteriol

103

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In the plant pathogen Pseudomonas syringae pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC. In this study, lscB and lscC were cloned from a genomic library of strain PG4180. Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene. lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC. Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180. The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC. Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P lac promoter. PCR screening in various P. syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P. syringae pathovars.The extracellular enzyme levansucrase (Lsc) (EC 2.4.1.10) catalyzes the following three reactions: (i) synthesis of levan from sucrose by transfructosylation while releasing glucose, (ii) hydrolysis of levan to monosaccharides of fructose, and (iii) exchange of [ 14 C]glucose in the reaction fructose-2,1-glucose plus [14 C]glucose to fructose-2,1-[ 14 C]glucose plus glucose (19). Levan is a high-molecular-weight ␤-(2,6)-polyfructan with extensive branching through ␤-(2,1) linkages. Lsc produces both linkage types.Levansucrases have been isolated from various bacteria, such as Bacillus subtilis, Streptococcus mutans, Zymomonas mobilis, Acetobacter diazotrophicus, Erwinia amylovora, and Pseudomonas syringae (2,9,18,22,34,41). In contrast to levansucrases from gram-positive bacteria, which differ widely in their biochemical characteristics, levansucrases from gramnegative bacteria are similar in their molecular mass and substrate-independent expression (16,22,23,43).The bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, causes formation of water-soaked lesions that develop into necrotic leaf spots surrounded by chlorotic halos. Like other exopolysaccharides (EPS), levan could have particular functions before or du...

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“…(12,107,189,195) and Zymomonas spp. (71,187,219,220), and, to a lesser extent, Lactobacillus spp. (196,211,213), Streptococcus spp.…”

Section: Fructansucrasesmentioning

confidence: 99%

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

Hijum

Kralj

Ozimek

et al. 2006

Microbiol Mol Biol Rev

391

315

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SUMMARY Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)8 barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.

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Nucleotide sequence of levansucrase gene (levU) of Zymomonas mobilis ZM1 (ATCC10988)

Cited by 57 publications

References 15 publications

A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

Characterization and Mutational Analysis of Three Allelic lsc Genes Encoding Levansucrase in Pseudomonas syringae

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

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