Suppressor mutations for crs mutants of Bacillus subtilis

1985 ◽  
Vol 31 (5) ◽  
pp. 429-435 ◽  
Author(s):  
D. Sun ◽  
I. Takahashi
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Resistance Gene ◽  
Resistance Mutation ◽  
Suppressor Mutation ◽  
Wild Type ◽  
Erythromycin Resistance ◽  
Wild Type Level ◽  
Suppressor Mutations ◽  
Metabolic Activities

Mutants of Bacillus subtilis which carried suppressor mutations for catabolite-resistance gene crsA47 were isolated from methylmethanesulfonate-treated cultures of GLU-47 (crsA47). The suppressor mutation, sca19, suppressed resistance of crsA47 mutant to glucose and other inhibitors of sporulation. Moreover, the suppressor mutation could restore the rate of growth and the level of IMP dehydrogenase and alkaline phosphatase of crsA47 mutant to the wild-type level. The sca19 mutation was also able to suppress catabolite resistance of other crs mutants. The map position of the sea19 mutation indicated that this mutation was an intergenic suppressor for the crs mutants. It was also found that an erythromycin-resistance mutation, ery1, could suppress the catabolite resistance of some of the crs mutants. Our results were discussed in relation to the importance of a proper state of metabolic activities and membrane functions during the initiation of sporulation.

scholarly journals Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Robert S. Brzozowski ◽  
Brooke R. Tomlinson ◽  
Michael D. Sacco ◽  
Judy J. Chen ◽  
Anika N. Ali ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Cell Size ◽  
Unknown Function ◽  
Suppressor Mutation ◽  
Gram Positive ◽  
Content Type ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

scholarly journals A Second-Site Mutation That Restores Replication of a Tat-Defective Human Immunodeficiency Virus

1999 ◽  
Vol 73 (4) ◽  
pp. 2781-2789 ◽  
Author(s):  
Koen Verhoef ◽  
Ben Berkhout
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Virus Replication ◽  
Suppressor Mutation ◽  
Large Set ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We previously constructed a large set of mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat with conservative amino acid substitutions in the activation domain. These Tat variants were analyzed in the context of the infectious virus, and several mutants were found to be defective for replication. In an attempt to obtain second-site suppressor mutations that could provide information on the Tat protein structure, some of the replication-impaired viruses were used as a parent for the isolation of revertant viruses with improved replication capacity. Sequence analysis of revertant viruses frequently revealed changes within thetat gene, most often first-site reversions either to the wild-type amino acid or to related amino acids that restore, at least partially, the Tat function and virus replication. Of 30 revertant cultures, we identified only one second-site suppressor mutation. The inactive Y26A mutant yielded the second-site suppressor mutation Y47N that partially restored trans-activation activity and virus replication. Surprisingly, when the suppressor mutation was introduced in the wild-type Tat background, it also improved thetrans-activation function of this protein about twofold. We conclude that the gain of function measured for the Y47N change is not specific for the Y26A mutant, arguing against a direct interaction of Tat amino acids 26 and 47 in the three-dimensional fold of this protein. Other revertant viruses did not contain any additional Tat changes, and some viruses revealed putative second-site Tat mutations that did not significantly improve Tat function and virus replication. We reason that these mutations were introduced by chance through founder effects or by linkage to suppressor mutations elsewhere in the virus genome. In conclusion, the forced evolution of mutant HIV-1 genomes, which is an efficient approach for the analysis of RNA regulatory motifs, seems less suited for the analysis of the structure of this small transcription factor, although protein variants with interesting properties can be generated.

Suppression of dual-effect mutants of the L-ribulokinase structural gene of Escherichia coli B/r

1971 ◽  
Vol 17 (8) ◽  
pp. 1105-1114
Author(s):  
Morad Abou-Sabe
Keyword(s):  
Enzyme Level ◽  
The Other ◽  
Wild Type ◽  
Dual Effect ◽  
Wild Type Level ◽  
Suppressor Mutations ◽  
Extragenic Suppressors ◽  
Escherichia Coli B ◽  
Distinct Increase ◽  
Arabinose Isomerase

A number of extragenic suppressors for two-dual effect, polarity, and hyperinducible L-ribulokinaseless mutants were isolated and enzymatically characterized. The effect of the suppressors on L-arabinose isomerase and L-ribulokinase enzyme activities was tested using a variety of arar suppressor-carrying strains. The effect of these suppressor mutations on T4 amber and ocher mutants was also studied. Results show that low-dual-effect, i.e. polarity, araB− suppressor-carrying strains invariably have a distinct increase in the L-arabinose isomerase enzyme level, above the wild-type level, irrespective of the restoration of the L-ribulokinase enzyme activity. High-dual-effect, hyperinducible, suppressed mutants, on the other hand, were not significantly altered in their L-arabinose isomerase enzyme level. It is also found that four different T4 amber mutants were suppressible by all tested suppressor-carrying strains, while T4 ocher mutants were not. It is concluded that the suppressor mutations studied are amber-like suppressors acting at the translation level, and that both polarity mutant araB68 and hyperinducible mutant araBA87 are nonsense mutants. The phenomenon of hyperinducibility is also discussed.

scholarly journals Genetic analysis of the phosphatases in Aspergillus nidulans

1965 ◽  
Vol 6 (1) ◽  
pp. 13-26 ◽  
Author(s):  
G. Dorn
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Genetic Analysis ◽  
Aspergillus Nidulans ◽  
Alkaline Ph ◽  
Wild Type ◽  
Linkage Groups ◽  
Partial Restoration ◽  
Suppressor Mutations ◽  
Alkaline And Acid Phosphatase

Summary1. A histochemical method has been applied to the detection of alkaline and acid phosphatase mutants in single colonies of Aspergillus nidulans.2. With the above method it has been possible to isolate mutants in which the alkaline and acid phosphatase activities are affected either separately or simultaneously.3. Crude extracts of wild-type A. nidulans contain four electrophoretically distinct phosphatase components, two with activity at alkaline pH and two with activity at acid pH. Genes affecting three of the four components have been identified.4. Two suppressor mutants of an alkaline phosphataseless mutant (palB7) have been isolated. In a strain carrying palB7 and one of these suppressors, the restoration of an alkaline phosphatase component is accompanied by loss of the faster acid phosphatase component. In a similar strain carrying the other suppressor, the partial restoration of the alkaline phosphatase component goes with an electrophoretic alteration of the slower acid phosphatase component.5. Genetic analysis of twenty-seven mutants has resulted in the identification of fifteen loci affecting the phosphatases. All these loci have been assigned to linkage groups, and twelve of them were also mapped meiotically in relation to other loci.6. One possible model (based on heteropolymeric proteins) has been proposed to account for the electrophoretic and genetic data on the various phosphatase and suppressor mutations.

scholarly journals Bacillus subtilis Phosphorylated PhoP: Direct Activation of the EσA- and Repression of the EσE-Responsive phoB-PS+V Promoters during Pho Response

2005 ◽  
Vol 187 (15) ◽  
pp. 5166-5178 ◽  
Author(s):  
Wael R. Abdel-Fattah ◽  
Yinghua Chen ◽  
Amr Eldakak ◽  
F. Marion Hulett
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pho Regulon ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphate Deprivation

ABSTRACT The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-PS, was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-PV. EσE was necessary and sufficient for PS expression in vitro. PS expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of σE is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP∼P) repressed PS in vitro via direct binding to the promoter, the first example of an EσE-responsive promoter that is repressed by PhoP∼P. Whereas either PhoP or PhoP∼P in the presence of EσA was sufficient to stimulate transcription from the phoB-PV promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP∼P were required for PV promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during Pi -replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

scholarly journals Alkaline Phosphatase Mutants of Bacillus subtilis

1975 ◽  
Vol 28 (3) ◽  
pp. 323 ◽  
Author(s):  
A RGlenn
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Control System ◽  
Phosphate Starvation ◽  
Wild Type ◽  
Alkaline Phosphatases ◽  
Wild Type Enzyme ◽  
Vegetative Cells ◽  
Specific Alkaline Phosphatase ◽  
Low Levels

Alkaline phosphatases from vegetative and sporulating cells of B. subtilis have been shown previously to be identical in all criteria examined. Despite this, 15 mutants producing low levels of the phosphatase during phosphate starvation of vegetative cells have been shown to produce high levels of the sporulation-specific alkaline phosphatase. It has been shown by imrnunochemical means that seven of these mutants when starved of phosphate produce low levels of normal wild-type enzyme. The sporulation form of the enzyme from one mutant (P-l00) has been shown to be identical with the phosphatases from vegetative and sporulating cells of the wild type. It is proposed that all the mutants have regulatory defects in the control of the alkaline phosphatase from vegetative cells but nevertheless retain an intact structural gene for the enzyme and the control system for the phosphatase during sporulation.

scholarly journals Suppressors of YpsA-mediated cell division inhibition in Bacillus subtilis

2020 ◽  
Author(s):  
Robert S. Brzozowski ◽  
Brooke R. Tomlinson ◽  
Michael D. Sacco ◽  
Judy J. Chen ◽  
Anika N. Ali ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Unknown Function ◽  
Suppressor Mutation ◽  
Bacterial Cell Division ◽  
Cell Width ◽  
Suppressor Mutations ◽  
Fundamental Biological Process ◽  
Critical Residues ◽  
Cell Division Inhibition

SUMMARYAlthough many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet to be discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, G132E) that rendered the mutated YpsA non-toxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation.GRAPHICAL ABSTRACTABBREVIATED SUMMARYIn Bacillus subtilis, we discovered that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. Analysis of one such suppressor mutation led us to a protein of unknown function, YfhS, which appears to play a role in regulating cell length and cell width.

scholarly journals The Bacillus subtilis yaaH Gene Is Transcribed by SigE RNA Polymerase during Sporulation, and Its Product Is Involved in Germination of Spores

1999 ◽  
Vol 181 (15) ◽  
pp. 4584-4591 ◽  
Author(s):  
Takeko Kodama ◽  
Hiromu Takamatsu ◽  
Kei Asai ◽  
Kazuo Kobayashi ◽  
Naotake Ogasawara ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Promoter Sequence ◽  
Wild Type ◽  
Erythromycin Resistance ◽  
Gene Encoding ◽  
Consensus Sequences ◽  
Protein Inactivation ◽  
Extension Analysis ◽  
Logarithmic Growth

ABSTRACT The expression of 21 novel genes located in the region fromdnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes,yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed thatyaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T 2) of sporulation in wild-type cells and in spoIIIG (SigG−) andspoIVCB (SigK−) mutants but not inspoIIAC (SigF−) and spoIIGAB(SigE−) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced fromT 2 of sporulation in a B. subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein. Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination ofyaaH mutant spores in a mixture ofl-asparagine, d-glucose,d-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective inl-alanine-stimulated germination. These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T 2 of sporulation and that it is required for thel-alanine-stimulated germination pathway.

scholarly journals The E1β and E2 Subunits of the Bacillus subtilis Pyruvate Dehydrogenase Complex Are Involved in Regulation of Sporulation

2002 ◽  
Vol 184 (10) ◽  
pp. 2780-2788 ◽  
Author(s):  
Haichun Gao ◽  
Xin Jiang ◽  
Kit Pogliano ◽  
Arthur I. Aronson
Keyword(s):  
Bacillus Subtilis ◽  
Enzymatic Activity ◽  
Pyruvate Dehydrogenase ◽  
Divalent Cations ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Decarboxylase ◽  
Stage Ii ◽  
Wild Type ◽  
Wild Type Level ◽  
Late Stages

ABSTRACT The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1α and E1β), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH). There are two promoters: one for the entire operon and an internal one in front of the pdhC gene. The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1α and E1β. Disruptions of the pdhB, pdhC, and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful, suggesting that E1α is essential. The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium. The pdhB and pdhC mutants sporulated to only 5% of the wild-type level, whereas the pdhD mutant strain sporulated to 55% of the wild-type level. This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to a function(s) of these subunits independent of enzymatic activity. Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations. Results from the expression of various spo-lacZ fusions revealed that the pdhB mutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas the pdhC mutant was blocked after the completion of engulfment (stage III). This analysis was confirmed by fluorescent membrane staining. The E1β and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation.

scholarly journals REGULATION OF PHOSPHATE METABOLISM IN NEUROSPORA CRASSA: ISOLATION OF MUTANTS DEFICIENT IN THE REPRESSIBLE ALKALINE PHOSPHATASE

Genetics ◽  
1974 ◽  
Vol 78 (2) ◽  
pp. 645-659
Author(s):  
Mary K Gleason ◽  
Robert L Metzenberg
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Linkage Group ◽  
Neurospora Crassa ◽  
Heat Stability ◽  
Phosphate Metabolism ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Group V ◽  
Suppressor Mutations

ABSTRACT Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pconc, and pregc. Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.

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