Cloning and characterization of a gene from  A1 involved in lipopolysaccharide biosynthesis

Potter, M; Lo, Reggie Y.C.

doi:10.1016/0378-1097(95)00140-z

Cited by 13 publications

(20 citation statements)

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“…Lgt2 has 55% similarity to the glycosyltransferases LgtB of Neisseria meningitidis and LpsA of Mannheimia haemolytica. These genes encode a ␤(1-4) galactosyltransferase involved in the addition of galactose to the ␣-chain of the N. meningitidis LOS molecule (41, 57, 60) and a glycosyltransferase involved in LPS core biosynthesis for M. haemolytica (42), respectively. Lgt3 has 28% similarity to LgtF of Haemophilus ducreyi and WaaE of Klebsiella pneumoniae.…”

Section: Resultsmentioning

confidence: 99%

Characterization of a Cluster of Three Glycosyltransferase Enzymes Essential for Moraxella catarrhalis Lipooligosaccharide Assembly

Edwards¹,

Allen

Gibson

et al. 2005

J Bacteriol

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Moraxella catarrhalis isolates express lipooligosaccharide (LOS) molecules on their surface, which share epitopes similar to that of the Neisseria and Haemophilus species. These common LOS epitopes have been implicated in various steps of pathogenesis for the different organisms. In this study, a cluster of three LOS glycosyltransferase genes (lgt) were identified in M. catarrhalis 7169, a strain that produces a serotype B LOS. Mutants in these glycosyltransferase genes were constructed, and the resulting LOS phenotypes were consistent with varying degrees of truncation compared to wild-type LOS. The LOS structures of each lgt mutant were no longer detected by a monoclonal antibody (MAb 4G5) specific to a highly conserved terminal epitope nor by a monoclonal antibody (MAb 3F7) specific to the serotype B LOS side chain. Mass spectrometry of the LOS glycoforms assembled by two of these lgt mutants indicated that lgt1 encodes an ␣(1-2) glucosyltransferase and the lgt2 encodes a ␤(1-4) galactosyltransferase. However, these structural studies could not delineate the function for lgt3. Therefore, M. catarrhalis lgt3 was introduced into a defined ␤(1-4) glucosyltransferase Haemophilus ducreyi 35000glu؊ mutant in trans, and monoclonal antibody analysis confirmed that Lgt3 complemented the LOS defect. These data suggest that lgt3 encodes a glucosyltransferase involved in the addition of a ␤(1-4)-linked glucose to the inner core. Furthermore, we conclude that this enzymatic step is essential for the assembly of the complete LOS glycoform expressed by M. catarrhalis 7169.Moraxella catarrhalis is a gram-negative human respiratory pathogen that causes 15 to 20% of acute otitis media in children (56). In addition, this bacterium is responsible for 10 to 35% of lower respiratory infections in adults with chronic obstructive pulmonary disease, the fourth leading cause of death in the United States (20). The emergence of M. catarrhalis as an important human pathogen has occurred in the past 15 years due to the increasing prevalence of ␤-lactamase-positive strains, the high incidence of recurrent infections despite successful antibiotic treatment, and the lack of an effective vaccine (10,39,50,56). Another factor that likely contributes to the persistence of M. catarrhalis disease is the lack of understanding of the basic bacterial factors and mechanisms that promote colonization and survival in the host.Although there have been a number of putative virulence factors described for M. catarrhalis, the actual role of these components in pathogenesis remains largely undefined (13,19,20,31,53). One of the most prominent surface components expressed in the outer membrane of this bacterium is the lipooligosaccharide (LOS) (12). The LOS of M. catarrhalis is similar to the lipopolysaccharide (LPS) of other gram-negative organisms, but it lacks a repeating O antigen (21). Previous studies have suggested that LOS is important for the pathogenesis of other respiratory pathogens, such as Neisseria meningitidis and Haemophilus influenzae, aid...

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Section: Resultsmentioning

confidence: 99%

Characterization of a Cluster of Three Glycosyltransferase Enzymes Essential for Moraxella catarrhalis Lipooligosaccharide Assembly

Edwards¹,

Allen

Gibson

et al. 2005

J Bacteriol

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“…(33), and Erwinia stewartii (27), the galE gene is found linked to genes involved in the synthesis of polysaccharides. In the third group of bacteria (e.g., Brucella abortus [63] and Pasteurella haemolytica [56]), the galE gene is linked to neither galactose metabolism genes nor polysaccharide genes. In addition, a few bacterial species have two functional galE genes.…”

Section: Discussionmentioning

confidence: 99%

The galE Gene of Campylobacter jejuni Is Involved in Lipopolysaccharide Synthesis and Virulence

et al. 2000

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Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS fromCampylobacter spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (wla) which is involved in the synthesis of the Campylobacter jejuni 81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with galE genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A Salmonella galE mutant was transformed with the galE gene from C. jejuni. The LPS analysis of wild-type, galE, and complemented galE Salmonella strains showed that the C. jejuni galE gene could restore the smooth wild-type Salmonella LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the galE gene from C. jejuni encoded this epimerase. We constructed a C. jejuni galE mutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the galE gene in the synthesis of C. jejuni LPS. The galE mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of C. jejuni is an important virulence factor.

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“…The amino acid sequence of β-1,4-galactosyltransferase of N. meningitidis showed homology to that of bacterial galactosyltransferases from Haemophilus influenza (High et al, 1993), Haemophilus ducreyi (Sun et al, 2000), Haemophilus sommnus (GenBank accession no. AF096997), and Pateurella haemolytica (Potter and Lo, 1995). All of these bacterial enzymes are involved in the biosynthesis of lipooligolysaccharide.…”

Section: Introductionmentioning

confidence: 99%

Expression and Characterization of β-1,4-Galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae

Park¹,

Lee²,

Il³

et al. 2002

BMB Reports

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The lgtB genes that encode β-1,4-galactosyltransferases from Neisseria meningitidis ATCC 13102 and gonorrhoeae ATCC 31151 were isolated by a polymerase chain reaction using the pfu DNA polymerase. They were expressed under the control of lac and T7 promoters in Escherichia coli M15 and BL21 (DE3). Although the genes were efficiently expressed in E. coli M15 at 37 o C (33 kDa), most of the β-1,4-galactosyltransferases that were produced were insoluble and proteolysed into enzymatically inactive polypeptides that lacked C-terminal residues (29.5 kDa and 28 kDa) during the purification steps. When the temperature of the cell growth was lowered to 25 o C, however, the solubility of the β-1,4-galactosyltransferases increased substantially. A stable N-terminal his-tagged recombinant enzyme preparation could be achieved with E. coli BL21 (DE3) that expressed lgtB. Therefore, the cloned β-1,4-galactosyltransferases were expressed under the control of the T7 promoter in E. coli BL21 (DE3), mostly to the soluble form at 25 o C. The proteins were easily purified to homogeneity by column chromatography using Ni-NTA resin, and were found to be active. The galactosyltransferases exhibited pH optimum at 6.5-7.0, and had an essential requirement for the Mn +2 ions for its action. The Mg +2 and Ca +2 ions showed about half of the galactosyltransferase activities with the Mn +2 ion. In the presence of the Fe +2 ion, partial activation was observed with the β-1,4-galactosyltransferase from N. meningitidis (64% of the enzyme activity with the Mn +2 ion), but not from N. gonorrhoeae. ions, the Mn +2 ion could not activate the enzyme activities. Also, the β-1,4-galactosyltransferase activity was 1.5-fold stimulated with the non-ionic detergent Triton X-100 (0.1-5%).

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Cloning and characterization of a gene from A1 involved in lipopolysaccharide biosynthesis

Cited by 13 publications

References 0 publications

Characterization of a Cluster of Three Glycosyltransferase Enzymes Essential for Moraxella catarrhalis Lipooligosaccharide Assembly

Characterization of a Cluster of Three Glycosyltransferase Enzymes Essential for Moraxella catarrhalis Lipooligosaccharide Assembly

The galE Gene of Campylobacter jejuni Is Involved in Lipopolysaccharide Synthesis and Virulence

Expression and Characterization of β-1,4-Galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae

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