scholarly journals Inactivation of the phosphoglucomutase gene pgm in Corynebacterium glutamicum affects cell shape and glycogen metabolism

2013 ◽  
Vol 33 (4) ◽  
Author(s):  
Gerd M. Seibold ◽  
Bernhard J. Eikmanns
Keyword(s):  
Cell Wall ◽  
Corynebacterium Glutamicum ◽  
Metabolic Pathways ◽  
Cell Shape ◽  
Glycogen Metabolism ◽  
Exponential Growth Phase ◽  
Constant Independent ◽  
Wild Type ◽  
P Content ◽  
Maltose Utilization

In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P. We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12% Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways.

scholarly journals Cardiolipin Alters Rhodobacter sphaeroides Cell Shape by Affecting Peptidoglycan Precursor Biosynthesis

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ti-Yu Lin ◽  
William S. Gross ◽  
George K. Auer ◽  
Douglas B. Weibel
Keyword(s):  
Cell Wall ◽  
Rhodobacter Sphaeroides ◽  
Cell Morphology ◽  
Cell Shape ◽  
Molecular Mechanisms ◽  
Wild Type ◽  
Anionic Phospholipid ◽  
Content Type ◽  
Lipid Ii ◽  
Topological Features

ABSTRACT Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG—a glycosyltransferase that performs the last step of lipid II biosynthesis—interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall. IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides. The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.

scholarly journals Vibrio choleraeadapts to sessile and motile lifestyles by cyclic di-GMP regulation of cell shape

2020 ◽  
Vol 117 (46) ◽  
pp. 29046-29054 ◽  
Author(s):  
Nicolas L. Fernandez ◽  
Brian Y. Hsueh ◽  
Nguyen T. Q. Nhu ◽  
Joshua L. Franklin ◽  
Yann S. Dufour ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Guanosine Monophosphate ◽  
Human Pathogen ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Regulatory Pathways ◽  
Matrix Components ◽  
Curved Rod

The cell morphology of rod-shaped bacteria is determined by the rigid net of peptidoglycan forming the cell wall. Alterations to the rod shape, such as the curved rod, occur through manipulating the process of cell wall synthesis. The human pathogenVibrio choleraetypically exists as a curved rod, but straight rods have been observed under certain conditions. While this appears to be a regulated process, the regulatory pathways controlling cell shape transitions inV. choleraeand the benefits of switching between rod and curved shape have not been determined. We demonstrate that cell shape inV. choleraeis regulated by the bacterial second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) by posttranscriptionally repressing expression ofcrvA, a gene encoding an intermediate filament-like protein necessary for curvature formation inV. cholerae.This regulation is mediated by the transcriptional cascade that also induces production of biofilm matrix components, indicating that cell shape is coregulated withV. cholerae’s induction of sessility. During microcolony formation, wild-typeV. choleraecells tended to exist as straight rods, while genetically engineering cells to maintain high curvature reduced microcolony formation and biofilm density. Conversely, straightV. choleraemutants have reduced swimming speed when using flagellar motility in liquid. Our results demonstrate regulation of cell shape in bacteria is a mechanism to increase fitness in planktonic and biofilm lifestyles.

scholarly journals VIBRIO CHOLERAE ADAPTS TO SESSILE AND MOTILE LIFESTYLES BY CYCLIC DI-GMP REGULATION OF CELL SHAPE

2020 ◽  
Author(s):  
Nicolas L. Fernandez ◽  
Nguyen T. Q. Nhu ◽  
Brian Y. Hsueh ◽  
Joshua L. Franklin ◽  
Yann S. Dufour ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Morphology ◽  
Cell Shape ◽  
Guanosine Monophosphate ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Regulatory Pathways ◽  
Matrix Components ◽  
Biofilm Matrix

AbstractThe cell morphology of rod-shaped bacteria is determined by the rigid net of peptidoglycan forming the cell wall. While V. cholerae grows into a curved shape under most conditions, straight rods have been observed. However, the signals and regulatory pathways controlling cell shape transitions in V. cholerae and the benefits of switching between rod and curved shape have not been determined. We demonstrate that cell shape in V. cholerae is regulated by the bacterial second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) by repressing expression of crvA, a gene encoding an intermediate filament-like protein necessary for curvature formation in V. cholerae. This regulation is mediated by the transcriptional cascade that also induces production of biofilm matrix components, indicating that cell shape is coregulated with V. cholerae’s induction of sessility. Wild-type V. cholerae cells adhering to a surface lose their characteristic curved shape to become as straight as cells lacking crvA while genetically engineering cells to maintain high curvature reduced microcolony formation and biofilm density. Conversely, straight V. cholerae mutants have reduced speed when swimming using flagellar motility in liquid. Our results demonstrate regulation of cell shape in bacteria is a mechanism to increase fitness in planktonic or biofilm lifestyles.

scholarly journals Metabolic Function of Corynebacterium glutamicum Aminotransferases AlaT and AvtA and Impact on l-Valine Production

2008 ◽  
Vol 74 (24) ◽  
pp. 7457-7462 ◽  
Author(s):  
Jan Marienhagen ◽  
Lothar Eggeling
Keyword(s):  
Corynebacterium Glutamicum ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Exponential Growth Phase ◽  
Dependent Manner ◽  
Wild Type ◽  
Growth Deficiency ◽  
In The Wild ◽  
Order Of Magnitude ◽  
Combined Data

ABSTRACT Aminotransferases (ATs) interacting with l-alanine are the least studied bacterial ATs. Whereas AlaT converts pyruvate to l-alanine in a glutamate-dependent reaction, AvtA is able to convert pyruvate to l-alanine in an l-valine-dependent manner. We show here that the wild type of Corynebacterium glutamicum with a deletion of either of the corresponding genes does not exhibit an explicit growth deficiency. However, a double mutant was auxotrophic for l-alanine, showing that both ATs can provide l-alanine and that they are the only ATs involved. Kinetic studies with isolated enzymes demonstrate that the catalytic efficiency, k cat/K m , of AlaT is higher than 1 order of magnitude in the direction of l-alanine formation (3.5 × 104 M−1 s−1), but no preference was apparent for AvtA, suggesting that AlaT is the principal l-alanine-supplying enzyme. This is in line with the cytosolic l-alanine concentration, which is reduced in the exponential growth phase from 95 mM to 18 mM by a deletion of alaT, whereas avtA deletion decreases the l-alanine concentration only to 76 mM. The combined data show that the presence of both ATs has subtle but obvious consequences on balancing intracellular amino acid pools in the wild type. The consequences are more obvious in an l-valine production strain where a high intracellular drain-off of the l-alanine precursor pyruvate prevails. We therefore used deletion of alaT to successfully reduce the contaminating l-alanine in extracellular accumulated l-valine by 80%.

scholarly journals Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

Microbiology ◽  
2009 ◽  
Vol 155 (2) ◽  
pp. 347-358 ◽  
Author(s):  
Gerd M. Seibold ◽  
Martin Wurst ◽  
Bernhard J. Eikmanns
Keyword(s):  
Corynebacterium Glutamicum ◽  
Glycogen Metabolism ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Glucose Phosphorylation ◽  
Maltose Utilization ◽  
Altered Cell ◽  
Maltose Phosphorylase ◽  
Maltodextrin Phosphorylase

Corynebacterium glutamicum transiently accumulates large amounts of glycogen, when cultivated on glucose and other sugars as a source of carbon and energy. Apart from the debranching enzyme GlgX, which is required for the formation of maltodextrins from glycogen, α-glucan phosphorylases were assumed to be involved in glycogen degradation, forming α-glucose 1-phosphate from glycogen and from maltodextrins. We show here that C. glutamicum in fact possesses two α-glucan phosphorylases, which act as a glycogen phosphorylase (GlgP) and as a maltodextrin phosphorylase (MalP). By chromosomal inactivation and subsequent analysis of the mutant, cg1479 was identified as the malP gene. The deletion mutant C. glutamicum ΔmalP completely lacked MalP activity and showed reduced intracellular glycogen degradation, confirming the proposed pathway for glycogen degradation in C. glutamicum via GlgP, GlgX and MalP. Surprisingly, the ΔmalP mutant showed impaired growth, reduced viability and altered cell morphology on maltose and accumulated much higher concentrations of glycogen and maltodextrins than the wild-type during growth on this substrate, suggesting an additional role of MalP in maltose metabolism of C. glutamicum. Further assessment of enzyme activities revealed the presence of 4-α-glucanotransferase (MalQ), glucokinase (Glk) and α-phosphoglucomutase (α-Pgm), and the absence of maltose hydrolase, maltose phosphorylase and β-Pgm, all three known to be involved in maltose utilization by Gram-positive bacteria. Based on these findings, we conclude that C. glutamicum metabolizes maltose via a pathway involving maltodextrin and glucose formation by MalQ, glucose phosphorylation by Glk and maltodextrin degradation via the reactions of MalP and α-Pgm, a pathway hitherto known to be present in Gram-negative rather than in Gram-positive bacteria.

scholarly journals PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum

2003 ◽  
Vol 185 (16) ◽  
pp. 4779-4786 ◽  
Author(s):  
Noelia Costa-Riu ◽  
Andreas Burkovski ◽  
Reinhard Krämer ◽  
Roland Benz
Keyword(s):  
Cell Wall ◽  
Corynebacterium Glutamicum ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Deletion Strain ◽  
Wild Type ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Reverse Transcription Pcr

ABSTRACT The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA. porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032ΔporA. Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA.

scholarly journals Different Modes of Diaminopimelate Synthesis and Their Role in Cell Wall Integrity: a Study withCorynebacterium glutamicum

1998 ◽  
Vol 180 (12) ◽  
pp. 3159-3165 ◽  
Author(s):  
Axel Wehrmann ◽  
Bodo Phillipp ◽  
Hermann Sahm ◽  
Lothar Eggeling
Keyword(s):  
Cell Wall ◽  
Mechanical Stress ◽  
Corynebacterium Glutamicum ◽  
Organic Nitrogen ◽  
Nitrogen Sources ◽  
Wild Type ◽  
Excess Carbon ◽  
Directed Mutation ◽  
High Affinity ◽  
Nitrogen Donor

ABSTRACT In eubacteria, there are three slightly different pathways for the synthesis of m-diaminopimelate (m-DAP), which is one of the key linking units of peptidoglycan. Surprisingly, for unknown reasons, some bacteria use two of these pathways together. An example isCorynebacterium glutamicum, which uses both the succinylase and dehydrogenase pathways for m-DAP synthesis. In this study, we clonedapD and prove by enzyme experiments that this gene encodes the succinylase (M r = 24082), initiating the succinylase pathway of m-DAP synthesis. By using gene-directed mutation, dapD, as well as dapE encoding the desuccinylase, was inactivated, thereby forcing C. glutamicum to use only the dehydrogenase pathway of m-DAP synthesis. The mutants are unable to grow on organic nitrogen sources. When supplied with low ammonium concentrations but excess carbon, their morphology is radically altered and they are less resistant to mechanical stress than the wild type. Since the succinylase has a high affinity toward its substrate and uses glutamate as the nitrogen donor, while the dehydrogenase has a low affinity and incorporates ammonium directly, the m-DAP synthesis is another example of twin activities present in bacteria for access to important metabolites such as the well-known twin activities for the synthesis of glutamate or for the uptake of potassium.

Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

1976 ◽  
Vol 34 ◽  
pp. 54-55
Author(s):  
Karen S. Howard ◽  
H. D. Braymer ◽  
M. D. Socolofsky ◽  
S. A. Milligan
Keyword(s):  
Cell Wall ◽  
Neurospora Crassa ◽  
Wild Type ◽  
Morphological Comparison ◽  
Small Areas ◽  
Solid Media ◽  
Thin Sectioning ◽  
X Cells ◽  
Mutant Cells ◽  
Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

scholarly journals Inhibition of cell expansion enhances cortical microtubule stability in the root apex of Arabidopsis thaliana

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Veronica Giourieva ◽  
Emmanuel Panteris
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Wall ◽  
Cell Expansion ◽  
Cortical Microtubule ◽  
Root Apex ◽  
Cellulose Synthase ◽  
Cellulose Biosynthesis ◽  
Cortical Microtubules ◽  
Wild Type ◽  
Microtubule Stability

Abstract Background Cortical microtubules regulate cell expansion by determining cellulose microfibril orientation in the root apex of Arabidopsis thaliana. While the regulation of cell wall properties by cortical microtubules is well studied, the data on the influence of cell wall to cortical microtubule organization and stability remain scarce. Studies on cellulose biosynthesis mutants revealed that cortical microtubules depend on Cellulose Synthase A (CESA) function and/or cell expansion. Furthermore, it has been reported that cortical microtubules in cellulose-deficient mutants are hypersensitive to oryzalin. In this work, the persistence of cortical microtubules against anti-microtubule treatment was thoroughly studied in the roots of several cesa mutants, namely thanatos, mre1, any1, prc1-1 and rsw1, and the Cellulose Synthase Interacting 1 protein (csi1) mutant pom2-4. In addition, various treatments with drugs affecting cell expansion were performed on wild-type roots. Whole mount tubulin immunolabeling was applied in the above roots and observations were performed by confocal microscopy. Results Cortical microtubules in all mutants showed statistically significant increased persistence against anti-microtubule drugs, compared to those of the wild-type. Furthermore, to examine if the enhanced stability of cortical microtubules was due to reduced cellulose biosynthesis or to suppression of cell expansion, treatments of wild-type roots with 2,6-dichlorobenzonitrile (DCB) and Congo red were performed. After these treatments, cortical microtubules appeared more resistant to oryzalin, than in the control. Conclusions According to these findings, it may be concluded that inhibition of cell expansion, irrespective of the cause, results in increased microtubule stability in A. thaliana root. In addition, cell expansion does not only rely on cortical microtubule orientation but also plays a regulatory role in microtubule dynamics, as well. Various hypotheses may explain the increased cortical microtubule stability under decreased cell expansion such as the role of cell wall sensors and the presence of less dynamic cortical microtubules.

scholarly journals Probing bacterial cell wall growth by tracing wall-anchored protein complexes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yi-Jen Sun ◽  
Fan Bai ◽  
An-Chi Luo ◽  
Xiang-Yu Zhuang ◽  
Tsai-Shun Lin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Bernoulli Shift ◽  
Protein Complexes ◽  
Axial Direction ◽  
Cell Wall Growth ◽  
Flagellar Motors ◽  
Shift Map ◽  
Dynamic Assembly ◽  
Wall Growth

AbstractThe dynamic assembly of the cell wall is key to the maintenance of cell shape during bacterial growth. Here, we present a method for the analysis of Escherichia coli cell wall growth at high spatial and temporal resolution, which is achieved by tracing the movement of fluorescently labeled cell wall-anchored flagellar motors. Using this method, we clearly identify the active and inert zones of cell wall growth during bacterial elongation. Within the active zone, the insertion of newly synthesized peptidoglycan occurs homogeneously in the axial direction without twisting of the cell body. Based on the measured parameters, we formulate a Bernoulli shift map model to predict the partitioning of cell wall-anchored proteins following cell division.

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