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MKAN27435 is required for the biosynthesis of higher subclasses of lipooligosaccharides in Mycobacterium kansasii.

Nataraj V, Pang PC, Haslam SM, Veerapen N, Minnikin DE, Dell A, Besra GS, Bhatt A - PLoS ONE (2015)

Bottom Line: Using Specialized Transduction, a phage-based gene knockout tool previously used to generate mutants in other mycobacteria, we generated a MKAN27435 mutant.The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII.Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.

ABSTRACT
Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate mutants in other mycobacteria, we generated a MKAN27435 mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.

No MeSH data available.


Related in: MedlinePlus

Autoradiograph of 2-D TLC analysis of polar lipids extracted from M. kansasii WT, ΔMKAN27435, ΔMKAN27435-C strains.Arrows indicate the different LOS species. I and II indicate system E dimension 1 and 2 respectively. Dimension I: Chloroform: Methanol: H2O (60:30:6); Dimension II: Chloroform: Acetic acid: Methanol: H2O (40:25:3:6).
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pone.0122804.g002: Autoradiograph of 2-D TLC analysis of polar lipids extracted from M. kansasii WT, ΔMKAN27435, ΔMKAN27435-C strains.Arrows indicate the different LOS species. I and II indicate system E dimension 1 and 2 respectively. Dimension I: Chloroform: Methanol: H2O (60:30:6); Dimension II: Chloroform: Acetic acid: Methanol: H2O (40:25:3:6).

Mentions: Given the presence of MKAN27435 in a putative LOS biosynthesis cluster, and the outcomes of the bioinformatics analysis described above, we sought to probe the role of MKAN27435 as a GTF involved in the biosynthesis of LOS’s in M. kansasii. First, a mutant of MKAN27435 was generated in the parental M. kansasii ATCC12478 strain (referred to as M. kansasii WT henceforth) using Specialized Transduction. Next, cultures of the strains were grown in the presence of [14C]-acetate to label lipids, and LOS’s were isolated as part of the polar lipid fraction from the labeled cultures. Separation of the polar lipids by 2D TLC [25] revealed an altered pattern of LOS species in the mutant strain; while M. kansasii WT produced all the subclasses of LOS’s (LOS-I, LOS-II, LOS-III, LOS-IV, LOS-V, LOS-VI and LOS-VII) the mutant produced LOS-I, LOS-II and LOS-III, but LOS-IV, LOS-V, LOS-VI and LOS-VII were missing in the strain (Fig 2). Furthermore, a new set of less abundant polar lipid species were detected in the mutant strain (Fig 2). Synthesis of all LOS species was restored in the mutant strain on introduction of a plasmid-encoded copy of MKAN27435 (Fig 2), indicating that the observed alteration in LOS patterns in the mutant was solely due to the loss of MKAN27435 function. In addition to the above phenotypes, we did observe a faint spot migrating closest to the origin in the 2D-TLC plate for the M. kansasii WT strain, which was absent in the mutant strain. However, as the spot did not reappear in the complemented strain, we presumed that this was an artifact and was unrelated to MKAN27435 function.


MKAN27435 is required for the biosynthesis of higher subclasses of lipooligosaccharides in Mycobacterium kansasii.

Nataraj V, Pang PC, Haslam SM, Veerapen N, Minnikin DE, Dell A, Besra GS, Bhatt A - PLoS ONE (2015)

Autoradiograph of 2-D TLC analysis of polar lipids extracted from M. kansasii WT, ΔMKAN27435, ΔMKAN27435-C strains.Arrows indicate the different LOS species. I and II indicate system E dimension 1 and 2 respectively. Dimension I: Chloroform: Methanol: H2O (60:30:6); Dimension II: Chloroform: Acetic acid: Methanol: H2O (40:25:3:6).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403928&req=5

pone.0122804.g002: Autoradiograph of 2-D TLC analysis of polar lipids extracted from M. kansasii WT, ΔMKAN27435, ΔMKAN27435-C strains.Arrows indicate the different LOS species. I and II indicate system E dimension 1 and 2 respectively. Dimension I: Chloroform: Methanol: H2O (60:30:6); Dimension II: Chloroform: Acetic acid: Methanol: H2O (40:25:3:6).
Mentions: Given the presence of MKAN27435 in a putative LOS biosynthesis cluster, and the outcomes of the bioinformatics analysis described above, we sought to probe the role of MKAN27435 as a GTF involved in the biosynthesis of LOS’s in M. kansasii. First, a mutant of MKAN27435 was generated in the parental M. kansasii ATCC12478 strain (referred to as M. kansasii WT henceforth) using Specialized Transduction. Next, cultures of the strains were grown in the presence of [14C]-acetate to label lipids, and LOS’s were isolated as part of the polar lipid fraction from the labeled cultures. Separation of the polar lipids by 2D TLC [25] revealed an altered pattern of LOS species in the mutant strain; while M. kansasii WT produced all the subclasses of LOS’s (LOS-I, LOS-II, LOS-III, LOS-IV, LOS-V, LOS-VI and LOS-VII) the mutant produced LOS-I, LOS-II and LOS-III, but LOS-IV, LOS-V, LOS-VI and LOS-VII were missing in the strain (Fig 2). Furthermore, a new set of less abundant polar lipid species were detected in the mutant strain (Fig 2). Synthesis of all LOS species was restored in the mutant strain on introduction of a plasmid-encoded copy of MKAN27435 (Fig 2), indicating that the observed alteration in LOS patterns in the mutant was solely due to the loss of MKAN27435 function. In addition to the above phenotypes, we did observe a faint spot migrating closest to the origin in the 2D-TLC plate for the M. kansasii WT strain, which was absent in the mutant strain. However, as the spot did not reappear in the complemented strain, we presumed that this was an artifact and was unrelated to MKAN27435 function.

Bottom Line: Using Specialized Transduction, a phage-based gene knockout tool previously used to generate mutants in other mycobacteria, we generated a MKAN27435 mutant.The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII.Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.

ABSTRACT
Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate mutants in other mycobacteria, we generated a MKAN27435 mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.

No MeSH data available.


Related in: MedlinePlus