Fluorescent Phospholipid Analogs as Microscopic Probes for Detection of the Mycolic Acid-Containing Layer in<i>Corynebacterium glutamicum</i>: Detecting Alterations in the Mycolic Acid-Containing Layer Following Ethambutol Treatment

Kumagai, Yutaro; Hirasawa, Takashi; Hayakawa, Kenshi; Nagai, Kazuo; Wachi, Masaaki

doi:10.1271/bbb.69.2051

Cited by 14 publications

(20 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was previously reported that fDHPE stains a mycolate layer and Nile Red stains cytoplasmic membrane in C. glutamicum (19). Mycolate layer formation is known to occur after cytoplasmic membrane and peptidoglycan layer formation (19). Both Nile Red and fDHPE stained the septum in both ⌬cgR_1596 and ⌬cgR_ 1596-cgR_2070 (Fig.…”

Section: Vol 190 2008 Genes Involved In Cell Separation In C Glutamentioning

confidence: 77%

“…To confirm whether ⌬cgR_1596 and ⌬cgR_1596-cgR_ 2070 are impaired in septum formation or cell separation, fDHPE and Nile Red staining were tested. It was previously reported that fDHPE stains a mycolate layer and Nile Red stains cytoplasmic membrane in C. glutamicum (19). Mycolate layer formation is known to occur after cytoplasmic membrane and peptidoglycan layer formation (19).…”

Section: Vol 190 2008 Genes Involved In Cell Separation In C Glutamentioning

confidence: 98%

See 1 more Smart Citation

Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R

et al. 2008

View full text Add to dashboard Cite

In previous work, random genome deletion mutants of Corynebacterium glutamicum R were generated using the insertion sequence (IS) element IS31831 and the Cre/loxP excision system. One of these mutants, C. glutamicum strain RD41, resulting from the deletion of a 10.1-kb genomic region (⌬cgR_1595 through cgR_ 1604) from the WT strain, showed cell elongation, and several lines appeared on the cell surface (bamboo shape). The morphological changes were suppressed by overexpression of cgR_1596. Single disruption of cgR_ 1596 in WT C. glutamicum R resulted in morphological changes similar to those observed in the RD41 strain. CgR_1596 has a predicted secretion signal peptide in the amino-terminal region and a predicted NlpC/P60 domain, which is conserved in cell wall hydrolases, in the carboxyl-terminal region. In C. glutamicum R, CgR_ 0802, CgR_1596, CgR_2069, and CgR_2070 have the NlpC/P60 domain; however, only simultaneous disruption of cgR_1596 and cgR_2070, and not cgR_2070 single disruption, resulted in cell growth delay and more severe morphological changes than disruption of cgR_1596. Transmission electron microscopy revealed multiple septa within individual cells of cgR_1596 single and cgR_1596-cgR_2070 double disruptants. Taken together, these results suggest that cgR_1596 and cgR_2070 are involved in cell separation and cell growth in C. glutamicum.The gram-positive, high-GC-content bacterium Corynebacterium glutamicum has been one of the most important bacteria in industry for several decades due to its high production of amino acids such as glutamate (16). The bacterium often shows a V-shaped cell form under the microscope, and its coryneform rod-shaped cells have one side of their poles slightly wider than the other. Since its unique characteristics enable it to produce such large amounts of amino acids, most C. glutamicum research has remained focused on the creation of highly productive strains on the one hand and analysis of metabolic flow regulation on the other (14,15,32). Consequently, the unusual behavior of the bacterium to produce V-shaped cells has elicited less research attention. The cell division system resulting in V-shaped cells is known as snapping division (37). Besides C. glutamicum, other Actinomycetales such as Arthorobacter, Nocardia, and Mycobacteria are known to produce V-shaped cells (9,18,28,38). Although several genes involved in cell division (13,17,20,30,34,47) and cell morphology (21,35,44,45) in C. glutamicum have been characterized, the molecular mechanism of the snapping division is still largely unknown.Cell division is achieved by the consecutive actions of cell extension, chromosome replication and segregation, and cell separation. In most prokaryotic and eukaryotic species, cell separation starts by constriction and the subsequent formation of two equivalent daughter cells. After completion of chromosome replication and segregation of the daughter chromosomes to the two halves of the cell, constriction occurs at a predetermined site and two progeny cells are produced. ...

show abstract

Section: Vol 190 2008 Genes Involved In Cell Separation In C Glutamentioning

confidence: 77%

Section: Vol 190 2008 Genes Involved In Cell Separation In C Glutamentioning

confidence: 98%

Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Figure 3B shows the results of this automated image analysis for DivIVA-mCherry-expressing C. glutamicum cells (left side, cells without EMB treatment). The top, middle, and bottom kymographs represent DivIVA-mCherry localization, azido- d -alanine labeling, and 2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine (DHPE) fluorescence distribution (representing MA distribution) ( 32 ), respectively. Bio-orthogonal labeling with azido- d -alanine is specific for nascent PG synthesis, while DHPE staining does not discriminate between newly synthesized and old MAs.…”

Section: Resultsmentioning

confidence: 99%

“…Treatment with EMB was shown to mimic these phenotypes ( 34 ). The loss of apical mycolates has been demonstrated with the MA-specific dye DHPE, suggesting that the mycolate layer is added at the cell poles ( 32 ). Our data extend these investigations and show that EMB treatment specifically blocks elongation growth.…”

Section: Discussionmentioning

confidence: 99%

The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria

Schubert

Sieger

Giacomelli

et al. 2017

mBio

View full text Add to dashboard Cite

Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked; this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome).

show abstract

“…Methods commonly applied to increase cell wall permeability include the addition of detergents or chemicals, biotin limitation, and a temperature upshift. Although these rather unspecific methods have different points of contact, they all affect the mycolate layer by changing its lipid composition or fluidity, which indicates that the corynebacterial outer membrane represents a barrier to the flux of solutes (15,18,28,44). A deeper knowledge of the porins of mycolata that establish the natural main passage across the outermost membrane could make such treatments unnecessary and could presumably be a great economic advantage.…”

mentioning

confidence: 99%

Reconstitution Experiments and Gene Deletions Reveal the Existence of Two-Component Major Cell Wall Channels in the Genus Corynebacterium

et al. 2010

View full text Add to dashboard Cite

Two small polypeptides, PorA and PorH, are known to form cell wall channels in Corynebacterium glutamicum and in Corynebacterium efficiens. The genes coding for both polypeptides are localized in close proximity to one another between the genes coding for GroEl2 and a polyphosphate kinase (PKK2). In this study, we investigated the relationship of PorA and PorH to one another. The results suggested that the major cell wall channels of Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium diphtheriae need the obligatory presence of two distinct polypeptides, one of class PorA and one of class PorH, to form an active cell wall channel. Identification of genes coding for homologous proteins in the chromosome of Corynebacterium callunae suggested a similar result for this strain. Contrary to our previous reports on channel-forming proteins in these strains, a heterooligomeric structure composed of PorA and PorH is needed in all of them to form the major cell wall channel. This was concluded from complementation experiments using a porH-and porAdeficient C. glutamicum strain. The stringent necessity of proteins of either class to recover the wild-type channels was demonstrated by black lipid bilayer experiments using detergent or organic solvent extracts of the complemented porH-and porA-deficient C. glutamicum strain. The channel-forming capability of recombinant expressed, affinity-purified PorA and PorH proteins of C. glutamicum revealed that the channels consisted solely of these two components. This agreed with results obtained from a transcript coding for both channelforming components identified in C. glutamicum by Northern blot analysis and reverse transcription-PCR analysis. The transcription start point of the genes was determined by the rapid amplification of cDNA ends approach, allowing the prediction of the ؊35 and ؊10 regions of the promoter. The results demonstrate that the cell wall channels within the genus Corynebacterium may be formed by two-component oligomers.

show abstract

Fluorescent Phospholipid Analogs as Microscopic Probes for Detection of the Mycolic Acid-Containing Layer inCorynebacterium glutamicum: Detecting Alterations in the Mycolic Acid-Containing Layer Following Ethambutol Treatment

Cited by 14 publications

References 33 publications

Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R

Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R

The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria

Reconstitution Experiments and Gene Deletions Reveal the Existence of Two-Component Major Cell Wall Channels in the Genus Corynebacterium

Contact Info

Product

Resources

About