Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA

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.

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Dominant suppressors of yeast actin mutations that are reciprocally suppressed.

Genetics ◽

10.1093/genetics/121.4.675 ◽

1989 ◽

Vol 121 (4) ◽

pp. 675-683

Author(s):

A E Adams ◽

D Botstein

Keyword(s):

High Temperature ◽

Linkage Group ◽

Growth Defect ◽

Temperature Sensitive ◽

Mutant Library ◽

Suppressor Mutations ◽

Extragenic Suppressor ◽

New Gene ◽

Allele Specificity

Abstract A gene whose product is likely to interact with yeast actin was identified by the isolation of pseudorevertants carrying dominant suppressors of the temperature-sensitive (Ts) act1-1 mutation. Of 30 independent revertants analyzed, 29 were found to carry extragenic suppressor mutations and of these, 24/24 tested were found to be linked to each other. This linkage group identifies a new gene SAC6, whose product, by several genetic criteria, is likely to interact intimately with actin. First, although act1-1 sac6 strains are temperature-independent (Ts+), 4/17 sac6 mutant alleles tested are Ts in an ACT1+ background. Moreover, four Ts+ pseudorevertants of these ACT1+ sac6 mutants carry suppressor mutations in ACT1; significantly, three of these are again Ts in a SAC6+ background, and are most likely new act1 mutant alleles. Thus, mutations in ACT1 and SAC6 can suppress each other's defects. Second, sac6 mutations can suppress the Ts defects of the act1-1 and act1-2, but not act1-4, mutations. This allele specificity indicates the sac6 mutations do not suppress by simply bypassing the function of actin at high temperature. Third, act1-4 sac6 strains have a growth defect greater than that due to either of the single mutations alone, again suggesting an interaction between the two proteins. The mutant sac6 gene was cloned on the basis of dominant suppression from an act1-1 sac6 mutant library, and was then mapped to chromosome IV, less than 2 cM from ARO1.

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Two classes of extragenic suppressor mutations identify functionally distinct regions of the GroEL chaperone of Escherichia coli.

Journal of Bacteriology ◽

10.1128/jb.176.21.6558-6565.1994 ◽

1994 ◽

Vol 176 (21) ◽

pp. 6558-6565 ◽

Cited By ~ 15

Author(s):

J Zeilstra-Ryalls ◽

O Fayet ◽

C Georgopoulos

Keyword(s):

Escherichia Coli ◽

Suppressor Mutations ◽

Extragenic Suppressor

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Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function

Journal of Virology ◽

10.1128/jvi.72.4.2825-2831.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 2825-2831 ◽

Cited By ~ 14

Author(s):

Ioannis Tubulekas ◽

Peter Liljeström

Keyword(s):

Cleavage Site ◽

Semliki Forest Virus ◽

Virus Entry ◽

Hot Spot ◽

Structure And Function ◽

Vector System ◽

Lower Probability ◽

Suppressor Mutations ◽

And Function ◽

New Mutations

ABSTRACT The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.

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Intragenic suppressor mutations of the COQ8 protein kinase homolog restore coenzyme Q biosynthesis and function in Saccharomyces cerevisiae

PLoS ONE ◽

10.1371/journal.pone.0234192 ◽

2020 ◽

Vol 15 (6) ◽

pp. e0234192

Author(s):

Agape M. Awad ◽

Anish Nag ◽

Nguyen V. B. Pham ◽

Michelle C. Bradley ◽

Nour Jabassini ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Coenzyme Q ◽

Suppressor Mutations ◽

And Function ◽

Intragenic Suppressor

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Extragenic suppressor mutations that restore twitching motility to fimL mutants of P seudomonas aeruginosa are associated with elevated intracellular cyclic AMP levels

MicrobiologyOpen ◽

10.1002/mbo3.49 ◽

2012 ◽

Vol 1 (4) ◽

pp. 490-501 ◽

Cited By ~ 12

Author(s):

Laura M. Nolan ◽

Scott A. Beatson ◽

Larry Croft ◽

Peter M. Jones ◽

Anthony M. George ◽

...

Keyword(s):

Cyclic Amp ◽

Twitching Motility ◽

Suppressor Mutations ◽

Extragenic Suppressor

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Novel Insights into Conformational Rearrangements of the Bacterial Flagellar Switch Complex

mBio ◽

10.1128/mbio.00079-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 13

Author(s):

Tomofumi Sakai ◽

Tomoko Miyata ◽

Naoya Terahara ◽

Koichiro Mori ◽

Yumi Inoue ◽

...

Keyword(s):

Proton Translocation ◽

Coupling Efficiency ◽

Structure And Function ◽

Proton Channel ◽

Flagellar Motor ◽

Wild Type ◽

Suppressor Mutations ◽

Type Motor ◽

And Function ◽

Intersubunit Interactions

ABSTRACTThe flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. TheSalmonellaFliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCEThe bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.

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Sequence analysis of mutations arising during prolonged starvation of Salmonella typhimurium.

Genetics ◽

10.1093/genetics/132.2.303 ◽

1992 ◽

Vol 132 (2) ◽

pp. 303-310

Author(s):

M J Prival ◽

T A Cebula

Keyword(s):

Sequence Analysis ◽

Salmonella Typhimurium ◽

Dna Sequences ◽

Agar Medium ◽

Base Pairs ◽

Suppressor Mutations ◽

Extragenic Suppressor ◽

Mutant Site ◽

Extragenic Suppression ◽

Extragenic Suppressors

Abstract We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.

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Genetic Analysis of Flagellar Length Control in Chlamydomonas reinhardtii: A New Long-Flagella Locus and Extragenic Suppressor Mutations

Genetics ◽

10.1093/genetics/148.2.693 ◽

1998 ◽

Vol 148 (2) ◽

pp. 693-702

Author(s):

Catherine M Asleson ◽

Paul A Lefebvre

Keyword(s):

Genetic Analysis ◽

Chlamydomonas Reinhardtii ◽

Double Mutant ◽

Wild Type ◽

Suppressor Mutations ◽

Extragenic Suppressor ◽

Flagellar Regeneration

Abstract Flagellar length in the biflagellate alga Chlamydomonas reinhardtii is under constant and tight regulation. A number of mutants with defects in flagellar length control have been previously identified. Mutations in the three long-flagella (lf) loci result in flagella that are up to three times longer than wild-type length. In this article, we describe the isolation of long-flagellar mutants caused by mutations in a new LF locus, LF4. lf4 mutations were shown to be epistatic to lf1, while lf2 was found to be epistatic to lf4 with regard to the flagellar regeneration defect. Mutations in lf4 were able to suppress the synthetic flagella-less phenotype of the lf1, lf2 double mutant. In addition, we have isolated four extragenic suppressor mutations that suppress the long-flagella phenotype of lf1, lf2, or lf3 double mutants.

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C. ELEGANS unc-105 MUTATIONS AFFECT MUSCLE AND ARE SUPPRESSED BY OTHER MUTATIONS THAT AFFECT MUSCLE

Genetics ◽

10.1093/genetics/113.4.853 ◽

1986 ◽

Vol 113 (4) ◽

pp. 853-867

Author(s):

Eun-Chung Park ◽

H Robert Horvitz

Keyword(s):

Structure And Function ◽

Loss Of Function ◽

Wild Type ◽

Nematode Caenorhabditis Elegans ◽

High Frequencies ◽

C Elegans ◽

Wild Type Phenotype ◽

Extragenic Suppressor ◽

And Function ◽

And Behavior

ABSTRACT Certain mutations in the unc-105 II gene of the nematode Caenorhabditis elegans have dominant effects on morphology and behavior: animals become small, severely hypercontracted and paralyzed. These unc-105 mutants revert both spontaneously and with mutagens at high frequencies to a wild-type phenotype. Most of the reversion events are intragenic, apparently because the null (loss-of-function) phenotype of unc-105 is wild type. One revertant defined an extragenic suppressor locus, sup-20 X. Such suppressor alleles of sup-20 are rare, and the apparent null phenotype of sup-20 is embryonic lethality. By constructing animals genetically mosaic for sup-20, we have shown that the primary effect of sup-20 is in muscle cells. In addition to mutations in sup-20, other mutations causing muscle defects, such as unc-54 and unc-22 mutations, suppress the hypercontracted phenotype of unc-105. The ease of identifying nonhypercontracted revertants of unc-105 mutants greatly facilitates the isolation of new mutants defective in muscle structure and function.

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