scholarly journals Suppression of a Thermosensitive zipA Cell Division Mutant by Altering Amino Acid Metabolism

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Daniel E. Vega ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Cytoplasmic Membrane ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Cell Division Protein

ABSTRACTZipA is essential for cell division inEscherichia coli, acting early in the process to anchor polymers of FtsZ to the cytoplasmic membrane. Along with FtsA, FtsZ and ZipA form a proto-ring at midcell that recruits additional proteins to eventually build the division septum. Cells carrying the thermosensitivezipA1allele divide fairly normally at 30°C in rich medium but cease dividing at temperatures above 34°C, forming long filaments. In a search for suppressors of thezipA1allele, we found that deletions of specific genes involved in amino acid biosynthesis could partially rescue cell growth and division at 34°C or 37°C but not at 42°C. Notably, although a diverse group of amino acid biosynthesis gene deletions could partially rescue the growth ofzipA1cells at 34°C, only deletions of genes related to the biosynthesis of threonine, glycine, serine, and methionine could rescue growth at 37°C. Adding exogenous pyridoxal 5-phosphate (PLP), a cofactor for many of the enzymes affected by this study, partially suppressedzipA1mutant thermosensitivity. For many of the deletions, PLP had an additive rescuing effect on thezipA1mutant. Moreover, added PLP partially suppressed the thermosensitivity offtsQandftsKmutants and weakly suppressed anftsImutant, but it failed to suppressftsAorftsZthermosensitive mutants. Along with the ability of a deletion ofmetCto partially suppress theftsKmutant, our results suggest that perturbations of amino acid metabolic pathways, particularly those that redirect the flow of carbon away from the synthesis of threonine, glycine, or methionine, are able to partially rescue some cell division defects.IMPORTANCECell division of bacteria, such asEscherichia coli, is essential for their successful colonization. It is becoming increasingly clear that nutritional status and central metabolism can affect bacterial size and shape; for example, a metabolic enzyme (OpgH) can moonlight as a regulator of FtsZ, an essential cell division protein. Here, we demonstrate a link between amino acid metabolism and ZipA, another essential cell division protein that binds directly to FtsZ and tethers it to the cytoplasmic membrane. Our evidence suggests that altering flux through the methionine-threonine-glycine-serine pathways and supplementing with the enzyme cofactor pyridoxal-5-phosphate can partially compensate for an otherwise lethal defect in ZipA, as well as several other cell division proteins.

scholarly journals PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of N-Acetyl-L-Glutamate Kinase by L-Arginine in Corynebacterium glutamicum

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Meijuan Xu ◽  
Mi Tang ◽  
Jiamin Chen ◽  
Taowei Yang ◽  
Xian Zhang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Corynebacterium Glutamicum ◽  
Feedback Inhibition ◽  
Amino Acid Biosynthesis ◽  
Arginine Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Pii Protein

ABSTRACT PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum. Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria. IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum. Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.

scholarly journals Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during β-Lactam Production

2019 ◽  
Vol 86 (3) ◽  
Author(s):  
Min Wu ◽  
Ciprian G. Crismaru ◽  
Oleksandr Salo ◽  
Roel A. L. Bovenberg ◽  
Arnold J. M. Driessen
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Strain Improvement ◽  
Tryptophan Synthase ◽  
Cysteine Biosynthesis ◽  
Penicillin Production ◽  
Content Type ◽  
Classical Strain ◽  
The Impact

ABSTRACT To produce high levels of β-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for β-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production. IMPORTANCE Penicillium rubens is an important industrial producer of β-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

scholarly journals Contribution of the FtsQ Transmembrane Segment to Localization to the Cell Division Site

2007 ◽  
Vol 189 (20) ◽  
pp. 7273-7280 ◽  
Author(s):  
Dirk-Jan Scheffers ◽  
Carine Robichon ◽  
Gert Jan Haan ◽  
Tanneke den Blaauwen ◽  
Gregory Koningstein ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Protein ◽  
Cytoplasmic Membrane ◽  
Central Component ◽  
Transmembrane Segment ◽  
Division Site ◽  
Periplasmic Domain ◽  
Cell Division Protein

ABSTRACT The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.

scholarly journals Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate inEscherichia coli

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Tomokazu Ito ◽  
Kana Yamamoto ◽  
Ran Hori ◽  
Ayako Yamauchi ◽  
Diana M. Downs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Biosynthetic Pathway ◽  
Acid Metabolism ◽  
Binding Protein ◽  
Protein Family ◽  
Pleiotropic Effects ◽  
Vitamin B ◽  
Content Type ◽  
E Coli

ABSTRACTEscherichia coliYggS (COG0325) is a member of the highly conserved pyridoxal 5′-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B6and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed inyggS-deficientE. coliwas caused by the pyridoxine 5′-phosphate (PNP)-dependent overproduction of Val, which is toxic toE. coli. The data suggest that theyggSmutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of theyggSmutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for ayggSmutant ofE. coli.IMPORTANCERecent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown thatyggSmutations inE. coliresult in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in theyggSmutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of theyggSmutant inE. coliare due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.

scholarly journals The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-CoutConfiguration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

2016 ◽  
Vol 198 (23) ◽  
pp. 3186-3199 ◽  
Author(s):  
Amit Pathania ◽  
Arvind Kumar Gupta ◽  
Swati Dubey ◽  
Balasubramanian Gopal ◽  
Abhijit A. Sardesai
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cytoplasmic Membrane ◽  
Basic Amino Acid ◽  
Intramolecular Interactions ◽  
Membrane Topology ◽  
Content Type ◽  
E Coli ◽  
Terminal Domain

ABSTRACTArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export ofl-arginine (Arg) inEscherichia coliandl-lysine (Lys) and Arg inCorynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane ofE. coli, we have employed a combination of cysteine accessibilityin situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Coutconfiguration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO functionin vivoand that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO functionin vivoand their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomerin vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.IMPORTANCEThe orthologous proteins LysE ofC. glutamicumand ArgO ofE. colifunction as exporters of the basic amino acidsl-arginine andl-lysine and the basic amino acidl-arginine, respectively, and LysE can functionally substitute for ArgO when expressed inE. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic andin silicostudies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

scholarly journals An Escherichia coli Mutant That Makes Exceptionally Long Cells

2015 ◽  
Vol 197 (8) ◽  
pp. 1507-1514 ◽  
Author(s):  
Ziad W. El-Hajj ◽  
Elaine B. Newman
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Luria Broth ◽  
Small Cells ◽  
Content Type ◽  
E Coli ◽  
Coli Mutant ◽  
Rich Media ◽  
Shaped Cell ◽  
Cell Division Protein

ABSTRACTAlthoughEscherichia coliis a very small (1- to 2-μm) rod-shaped cell, here we describe anE. colimutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 μm. Theseextremelyelongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division.IMPORTANCEEscherichia coliis usually a very small bacterium, 1 to 2 μm long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usualE. colicell.E. colifilaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects ofE. coliphysiology that are difficult to investigate with small cells.

Structure of Aspartate-β-semialdehyde Dehydrogenase from Escherichia coli, a Key Enzyme in the Aspartate Family of Amino Acid Biosynthesis

1999 ◽  
Vol 289 (4) ◽  
pp. 991-1002 ◽  
Author(s):  
Andrea Hadfield ◽  
Gitay Kryger ◽  
Jun Ouyang ◽  
Gregory A. Petsko ◽  
Dagmar Ringe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Semialdehyde Dehydrogenase ◽  
Key Enzyme ◽  
Aspartate Family
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