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

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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The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

Journal of Bacteriology ◽

10.1128/jb.00322-16 ◽

2016 ◽

Vol 198 (16) ◽

pp. 2263-2274 ◽

Cited By ~ 18

Author(s):

Ryan N. C. Buensuceso ◽

Ylan Nguyen ◽

Kun Zhang ◽

Martin Daniel-Ivad ◽

Seiji N. Sugiman-Marangos ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cyclic Amp ◽

Tetratricopeptide Repeat ◽

Twitching Motility ◽

Wild Type ◽

Inner Membrane ◽

Content Type ◽

Terminal Domain ◽

C Terminus ◽

Independent Functions

ABSTRACTFimV is aPseudomonas aeruginosainner membrane protein that regulates intracellular cyclic AMP (cAMP) levels—and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)—by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the “FimV C-terminal domain,” which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861–919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689failed to restore wild-type motility in afimVmutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions.IMPORTANCEFimV is important for twitching motility and cAMP-dependent virulence gene expression inP. aeruginosa. FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

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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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Cyclic AMP-Independent Control of Twitching Motility in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00188-17 ◽

2017 ◽

Vol 199 (16) ◽

Cited By ~ 13

Author(s):

Ryan N. C. Buensuceso ◽

Martin Daniel-Ivad ◽

Sara L. N. Kilmury ◽

Tiffany L. Leighton ◽

Hanjeong Harvey ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cyclic Amp ◽

Response Regulator ◽

Opportunistic Pathogen ◽

Regulatory Elements ◽

Twitching Motility ◽

Camp Phosphodiesterase ◽

Content Type ◽

Polar Localization ◽

Camp Levels

ABSTRACT FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds—lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB—pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility. IMPORTANCE Pseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements—including the PilSR two-component system—to cell poles for optimal function.

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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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Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA

BMC Microbiology ◽

10.1186/s12866-014-0336-x ◽

2014 ◽

Vol 14 (1) ◽

Cited By ~ 4

Author(s):

Cheryl N Miller ◽

Shaun P Steele ◽

Jason C Brunton ◽

Ronald J Jenkins ◽

Eric D LoVullo ◽

...

Keyword(s):

Suppressor Mutations ◽

Extragenic Suppressor ◽

And Function

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Extragenic suppressor mutations of a β-tubulin mutation in the basidiomyceteCoprinus cinereus: Isolation and genetic and biochemical analyses

Current Microbiology ◽

10.1007/bf02089414 ◽

1990 ◽

Vol 20 (4) ◽

pp. 223-228 ◽

Cited By ~ 8

Author(s):

Takashi Kamada ◽

Hiroko Hirami ◽

Tohru Sumiyoshi ◽

Shigeru Tanabe ◽

Tsuneo Takemaru

Keyword(s):

Suppressor Mutations ◽

Extragenic Suppressor ◽

Biochemical Analyses ◽

Β Tubulin

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Cytochemical Evidence for Presynatic β-Adrenergic Regulation of Secretion in the Developing Rat Submandibular Gland

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079590 ◽

1977 ◽

Vol 35 ◽

pp. 430-431

Author(s):

L.S. Cutler

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Submandibular Gland ◽

Neonatal Rat ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Parotid Glands ◽

Adrenergic Agonist ◽

Rat Submandibular Gland

Many studies previously have shown that the B-adrenergic agonist isoproterenol and the a-adrenergic agonist norepinephrine will stimulate secretion by the adult rat submandibular (SMG) and parotid glands. Recent data from several laboratories indicates that adrenergic agonists bind to specific receptors on the secretory cell surface and stimulate membrane associated adenylate cyclase activity which generates cyclic AMP. The production of cyclic AMP apparently initiates a cascade of events which culminates in exocytosis. During recent studies in our laboratory it was observed that the adenylate cyclase activity in plasma membrane fractions derived from the prenatal and early neonatal rat submandibular gland was retractile to stimulation by isoproterenol but was stimulated by norepinephrine. In addition, in vitro secretion studies indicated that these prenatal and neonatal glands would not secrete peroxidase in response to isoproterenol but would secrete in response to norepinephrine. In contrast to these in vitro observations, it has been shown that the injection of isoproterenol into the living newborn rat results in secretion of peroxidase by the SMG (1).

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