• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.84-89
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• 2005

Phosphomannomutase (PMM) is a key enzyme in prokaryotes and eukaryotes, which catalyzes the conversion of ${\alpha}$-D-mannose 6-phosphate to ${\alpha}$-D-mannose 1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for many metabolic pathways in the cells. We report here on the isolation of a gene from a genomic library of Sphingomonas chungbukensis DJ77, the pmmC gene encoding phosphomannomutase. The gene was cloned into E. coli expression vector, and the sequence was analyzed. The ribosomal binding site GGAAG lays 5 bp upstream of the ORF of 750 bp, which is initiated by ATG codon and terminated by TAG. The predicted sequence of the enzyme consists of 249 amino acids with a molecular mass of 27.4 kDa and showed $86.9\%$ similarity to that of eukaryotic phosphomannomutase after bioinformatical analyses with the conserved domain search of NCBI. The purified gene product revealed the activity of phosphomannomutase. In conclusion, we confirmed that pmmC gene encodes phosphomannomutase actually.

• Journal of Microbiology and Biotechnology
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• v.13 no.1
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• pp.149-154
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• 2003

Numerous virulence factors such as O antigen have been proposed to account for the fulminating and destructive nature of V. vulnificus infections. To better characterize the role of O antigen, a pmm gene encoding a phosphomannomutase was identified and cloned from V. vulnificus. The deduced amino acid sequence of the pmm was 42 to 71% similar to that reported from other Enterobacteriaceae. Functions of the pmm gene in virulence were assessed by the construction of an isogenic mutant, whose pmm gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of pmm resulted in a loss of more than 90% of phosphomannomutase, and reintroduction of recombinant pmm could complement the decrease of phosphomannomutase activity, indicating that the pmm gene encodes the phosphomannomutase of V. vulnificus. There was no difference in the $LD_50S$ of the wild-type and the pmm mutant in mice, but the $LD_50S$ observed by the mutant complemented with recombinant pmm were lower. Therefore, it appears that PMM is less important in the pathogenesis of V. vulnificus than would have been predicted by examining the effects of injecting purified LPS into animals, but it is not completely dispensable for virulence in mice.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1293-1298
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• 2018

Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1-phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and $50^{\circ}C$, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose-1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.355-362
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• 2016

The phosphomannomutase/phosphoglucomutase gene (pmm/pgm) of Vibrio anguillarum (the causative agent of fish vibriosis) was cloned, and the open reading frame corresponded to a protein with 446 amino acids. The pmm/pgm gene showed a significant degree of sequence homology with the previously reported genes from V. mimicus, V. vulnificus, V. splendidus, and V. harveyi, with 92.3%, 91.4%, 89.9%, and 89.9% amino acid identity, respectively. By reverse transcriptase-polymerase chain reaction, we found that the pmm/pgm gene was upregulated under cold stress condition. The PMM/PGM protein is known to catalyze the interconversion between mannose-1-phosphate and mannose-6-phosphate or glucose-1-phosphate and glucose-6-phosphate, which are important intermediates for lipopolysaccharide (LPS) biosynthesis. To confirm the role of PMM/PGM in the LPS biosynthetic pathway, we constructed a knock out mutant by homologous recombination. The respective LPSs were isolated from the V. anguillarum wild-type and mutant strains, and changes were compared by subjecting them to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on the different patterns of the LPSs, we expect the pmm/pgm gene to have an important role in LPS biosynthesis. The pmm/pgm-deficient mutant of V. anguillarum will contribute to further studies about the role of LPS in V. anguillarum pathogenesis.

• Journal of Microbiology and Biotechnology
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• v.22 no.10
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• pp.1324-1329
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• 2012

Phosphomannomutase (ManB) is involved in the biosynthesis of GDP-mannose, which is vital for numerous processes such as synthesis of carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins. Here, we discovered that a deletion mutant of manB (BG101) in Streptomyces coelicolor (S. coelicolor) showed higher sensitivity to bacteriostatic chloramphenicol (CM) than the wild-type strain (M145), along with decreased production of CM metabolites. Deletion of manB also decreased the mRNA expression level of drug efflux pumps (i.e., cmlR1 and cmlR2) in S. coelicolor, resulting in increased sensitivity to CM. This is the first report on changes in antibiotic sensitivity to CM by deletion of one glycolysis-related enzyme in S. coelicolor, and the results suggest different approaches for studying the antibiotic-resistant mechanism and its regulation.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.168-176
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• 2005

Vibrio vulnificus, one of the marine bacterial pathogens causing septicemia, was detected using molecular methods, namely, PCR and/or Southern hybridization, and real-time PCR. Extracted and purified total DNAs by using commercial kits were used as templates for PCR. Multiplex-PCR was conducted by employing three sets of primers for the genes, hemolysin (vvhA), phosphomannomutase (pmm), and metalloprotease (vvpE), for V vulnificus virulence. The presence of DMSO ($5\%$) and BSA ($0.1\%$) in PCR reaction mixture improved a detection efficiency by higher PCR band intensities. TaqMan real-time PCR was carried out by using gene segment of vvhA as a target. Detection limit of PCR/Southern hybridization without enrichments was to be around $10^2\;cells\;g^{-1}$ of sample. However, those three methods using the enrichment at $35^{\circ}C$ in APW showed high sensitivity ($2\~10\;cells\;g^{-1}$ of sediments). Highly sensitive detection of V vulnificus by real-time PCR was achieved within $5\~6$ hr, whereas the detection by PCR/Southern hybridization required about 36 hr. Thus, it was evident that real-time PCR is the most rapid and efficient method for detecting V vulnificus in tidal flat sediments.

• Genomics & Informatics
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• v.12 no.4
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• pp.268-275
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• 2014

The harshness of legionellosis differs from mild Pontiac fever to potentially fatal Legionnaire's disease. The increasing development of drug resistance against legionellosis has led to explore new novel drug targets. It has been found that phosphoglucosamine mutase, phosphomannomutase, and phosphoglyceromutase enzymes can be used as the most probable therapeutic drug targets through extensive data mining. Phosphoglucosamine mutase is involved in amino sugar and nucleotide sugar metabolism. The purpose of this study was to predict the potential target of that specific drug. For this, the 3D structure of phosphoglucosamine mutase of Legionella pneumophila (strain Paris) was determined by means of homology modeling through Phyre2 and refined by ModRefiner. Then, the designed model was evaluated with a structure validation program, for instance, PROCHECK, ERRAT, Verify3D, and QMEAN, for further structural analysis. Secondary structural features were determined through self-optimized prediction method with alignment (SOPMA) and interacting networks by STRING. Consequently, we performed molecular docking studies. The analytical result of PROCHECK showed that 95.0% of the residues are in the most favored region, 4.50% are in the additional allowed region and 0.50% are in the generously allowed region of the Ramachandran plot. Verify3D graph value indicates a score of 0.71 and 89.791, 1.11 for ERRAT and QMEAN respectively. Arg419, Thr414, Ser412, and Thr9 were found to dock the substrate for the most favorable binding of S-mercaptocysteine. However, these findings from this current study will pave the way for further extensive investigation of this enzyme in wet lab experiments and in that way assist drug design against legionellosis.