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

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.

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

Bioscience Reports ◽

10.1042/bsr20130076 ◽

2013 ◽

Vol 33 (4) ◽

Cited By ~ 10

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.

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

Journal of Bacteriology ◽

10.1128/jb.185.16.4779-4786.2003 ◽

2003 ◽

Vol 185 (16) ◽

pp. 4779-4786 ◽

Cited By ~ 29

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090257 ◽

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).

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

Journal of Biological Research-Thessaloniki ◽

10.1186/s40709-021-00143-8 ◽

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.

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The Craterostigma plantagineum protein kinase CpWAK1 interacts with pectin and integrates different environmental signals in the cell wall

Planta ◽

10.1007/s00425-021-03609-0 ◽

2021 ◽

Vol 253 (5) ◽

Author(s):

Peilei Chen ◽

Valentino Giarola ◽

Dorothea Bartels

Keyword(s):

Cell Wall ◽

Stress Responses ◽

Expression Profiles ◽

Phylogenetic Analyses ◽

Cell Wall Protein ◽

Biological Processes ◽

Environmental Signals ◽

High Affinity ◽

Craterostigma Plantagineum ◽

Receptor Kinases

Abstract Main conclusion The cell wall protein CpWAK1 interacts with pectin, participates in decoding cell wall signals, and induces different downstream responses. Abstract Cell wall-associated protein kinases (WAKs) are transmembrane receptor kinases. In the desiccation-tolerant resurrection plant Craterostigma plantagineum, CpWAK1 has been shown to be involved in stress responses and cell expansion by forming a complex with the C. plantagineum glycine-rich protein1 (CpGRP1). This prompted us to extend the studies of WAK genes in C. plantagineum. The phylogenetic analyses of WAKs from C. plantagineum and from other species suggest that these genes have been duplicated after species divergence. Expression profiles indicate that CpWAKs are involved in various biological processes, including dehydration-induced responses and SA- and JA-related reactions to pathogens and wounding. CpWAK1 shows a high affinity for “egg-box” pectin structures. ELISA assays revealed that the binding of CpWAKs to pectins is modulated by CpGRP1 and it depends on the apoplastic pH. The formation of CpWAK multimers is the prerequisite for the CpWAK–pectin binding. Different pectin extracts lead to opposite trends of CpWAK–pectin binding in the presence of Ca2+ at pH 8. These observations demonstrate that CpWAKs can potentially discriminate and integrate cell wall signals generated by diverse stimuli, in concert with other elements, such as CpGRP1, pHapo, Ca2+[apo], and via the formation of CpWAK multimers.

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Transcriptome and metabolome profiling provide insights into molecular mechanism of pseudostem elongation in banana

BMC Plant Biology ◽

10.1186/s12870-021-02899-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Guiming Deng ◽

Fangcheng Bi ◽

Jing Liu ◽

Weidi He ◽

Chunyu Li ◽

...

Keyword(s):

Cell Wall ◽

Cell Elongation ◽

Molecular Mechanisms ◽

Specific Gene ◽

Wild Type ◽

Banana Plant ◽

Cell Wall Modification ◽

Dwarf Cultivar ◽

Important Trait ◽

Metabolome Profiling

AbstractBackgroundBanana plant height is an important trait for horticultural practices and semi-dwarf cultivars show better resistance to damages by wind and rain. However, the molecular mechanisms controlling the pseudostem height remain poorly understood. Herein, we studied the molecular changes in the pseudostem of a semi-dwarf banana mutant Aifen No. 1 (Musaspp. Pisang Awak sub-group ABB) as compared to its wild-type dwarf cultivar using a combined transcriptome and metabolome approach.ResultsA total of 127 differentially expressed genes and 48 differentially accumulated metabolites were detected between the mutant and its wild type. Metabolites belonging to amino acid and its derivatives, flavonoids, lignans, coumarins, organic acids, and phenolic acids were up-regulated in the mutant. The transcriptome analysis showed the differential regulation of genes related to the gibberellin pathway, auxin transport, cell elongation, and cell wall modification. Based on the regulation of gibberellin and associated pathway-related genes, we discussed the involvement of gibberellins in pseudostem elongation in the mutant banana. Genes and metabolites associated with cell wall were explored and their involvement in cell extension is discussed.ConclusionsThe results suggest that gibberellins and associated pathways are possibly developing the observed semi-dwarf pseudostem phenotype together with cell elongation and cell wall modification. The findings increase the understanding of the mechanisms underlying banana stem height and provide new clues for further dissection of specific gene functions.

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Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

Nucleic Acids Research ◽

10.1093/nar/gkab041 ◽

2021 ◽

Author(s):

Amit Ketkar ◽

Lane Smith ◽

Callie Johnson ◽

Alyssa Richey ◽

Makayla Berry ◽

...

Keyword(s):

Amino Acids ◽

Mutation Frequency ◽

Control Sequence ◽

Wild Type ◽

Binding Properties ◽

High Affinity ◽

G4 Dna ◽

G Quadruplex ◽

Forward Mutagenesis

Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

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With an Ear up Against the Wall: An Update on Mechanoperception in Arabidopsis.

Plants ◽

10.3390/plants10081587 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1587

Author(s):

Sara Behnami ◽

Dario Bonetta

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Immune Responses ◽

Mechanical Stress ◽

Mechanosensitive Channels ◽

Compensatory Responses ◽

Cell Wall Damage ◽

Mechanical Signals ◽

Hormone Signalling

Cells interpret mechanical signals and adjust their physiology or development appropriately. In plants, the interface with the outside world is the cell wall, a structure that forms a continuum with the plasma membrane and the cytoskeleton. Mechanical stress from cell wall damage or deformation is interpreted to elicit compensatory responses, hormone signalling, or immune responses. Our understanding of how this is achieved is still evolving; however, we can refer to examples from animals and yeast where more of the details have been worked out. Here, we provide an update on this changing story with a focus on candidate mechanosensitive channels and plasma membrane-localized receptors.

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Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

The Journal of General Physiology ◽

10.1085/jgp.200308784 ◽

2003 ◽

Vol 122 (3) ◽

pp. 295-306 ◽

Cited By ~ 52

Author(s):

Sonia Traverso ◽

Laura Elia ◽

Michael Pusch

Keyword(s):

Chloride Channels ◽

Bound State ◽

Side Chain ◽

Pore Channel ◽

Kinetic Properties ◽

Wild Type ◽

High Affinity ◽

Critical Residue ◽

Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

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