Isolated Suppressor Mutations Research Articles

During heat stress in Myxococcus xanthus, the CdbS PilZ domain protein, in concert with two PilZ-DnaK chaperones, perturbs chromosome organization and accelerates cell death.

C-di-GMP is a bacterial second messenger that regulates diverse processes in response to environmental or cellular cues. The nucleoid-associated protein (NAP) CdbA in Myxococcus xanthus binds c-di-GMP and DNA in a mutually exclusive manner in vitro. CdbA is essential for viability, and CdbA depletion causes defects in chromosome organization, leading to a block in cell division and, ultimately, cell death. Most NAPs are not essential; therefore, to explore the paradoxical cdbA essentiality, we isolated suppressor mutations that restored cell viability without CdbA. Most mutations mapped to cdbS, which encodes a stand-alone c-di-GMP binding PilZ domain protein, and caused loss-of-function of cdbS. Cells lacking CdbA and CdbS or only CdbS were fully viable and had no defects in chromosome organization. CdbA depletion caused post-transcriptional upregulation of CdbS accumulation, and this CdbS over-accumulation was sufficient to disrupt chromosome organization and cause cell death. CdbA depletion also caused increased accumulation of CsdK1 and CsdK2, two unusual PilZ-DnaK chaperones. During CdbA depletion, CsdK1 and CsdK2, in turn, enabled the increased accumulation and toxicity of CdbS, likely by stabilizing CdbS. Moreover, we demonstrate that heat stress, possibly involving an increased cellular c-di-GMP concentration, induced the CdbA/CsdK1/CsdK2/CdbS system, causing a CsdK1- and CsdK2-dependent increase in CdbS accumulation. Thereby this system accelerates heat stress-induced chromosome mis-organization and cell death. Collectively, this work describes a unique system that contributes to regulated cell death in M. xanthus and suggests a link between c-di-GMP signaling and regulated cell death in bacteria.

Correction: The Zinc-Finger Protein SOP1 Is Required for a Subset of the Nuclear Exosome Functions in Arabidopsis

Correct gene expression requires tight RNA quality control both at transcriptional and post-transcriptional levels. Using a splicing-defective allele of PASTICCINO2 (PAS2), a gene essential for plant development, we isolated suppressor mutations modifying pas2-1 mRNA profiles and restoring wild-type growth. Three suppressor of pas2 (sop) mutations modified the degradation of mis-spliced pas2-1 mRNA species, allowing the synthesis of a functional protein. Cloning of the suppressor mutations identified the core subunit of the exosome SOP2/RRP4, the exosome nucleoplasmic cofactor SOP3/HEN2 and a novel zinc-finger protein SOP1 that colocalizes with HEN2 in nucleoplasmic foci. The three SOP proteins counteract post-transcriptional (trans)gene silencing (PTGS), which suggests that they all act in RNA quality control. In addition, sop1 mutants accumulate some, but not all of the misprocessed mRNAs and other types of RNAs that are observed in exosome mutants. Taken together, our data show that SOP1 is a new component of nuclear RNA surveillance that is required for the degradation of a specific subset of nuclear exosome targets.

The Zinc-Finger Protein SOP1 Is Required for a Subset of the Nuclear Exosome Functions in Arabidopsis.

Correct gene expression requires tight RNA quality control both at transcriptional and post-transcriptional levels. Using a splicing-defective allele of PASTICCINO2 (PAS2), a gene essential for plant development, we isolated suppressor mutations modifying pas2-1 mRNA profiles and restoring wild-type growth. Three suppressor of pas2 (sop) mutations modified the degradation of mis-spliced pas2-1 mRNA species, allowing the synthesis of a functional protein. Cloning of the suppressor mutations identified the core subunit of the exosome SOP2/RRP4, the exosome nucleoplasmic cofactor SOP3/HEN2 and a novel zinc-finger protein SOP1 that colocalizes with HEN2 in nucleoplasmic foci. The three SOP proteins counteract post-transcriptional (trans)gene silencing (PTGS), which suggests that they all act in RNA quality control. In addition, sop1 mutants accumulate some, but not all of the misprocessed mRNAs and other types of RNAs that are observed in exosome mutants. Taken together, our data show that SOP1 is a new component of nuclear RNA surveillance that is required for the degradation of a specific subset of nuclear exosome targets.

Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli.

Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell. Most bacteria possess multiple diguanylate cyclases and phosphodiesterases. Genetic studies have proposed that these enzymes show signaling specificity by contributing to distinct cellular processes without much cross talk. Thus, spatial separation of individual c-di-GMP signaling units was postulated. However, since most cyclases and phosphodiesterases harbor N-terminal signal input domains, it is equally possible that most of these enzymes lack their activating signals under laboratory conditions, thereby simulating signaling specificity on a genetic level. We demonstrate that a subset of E. coli phosphodiesterases can be activated genetically to affect the global c-di-GMP pool and thus influence different c-di-GMP-dependent processes. Although this does not exclude spatial confinement of individual phosphodiesterases, this study emphasizes the importance of environmental signals for activation of phosphodiesterases.

Constitutive Stringent Response Restores Viability of Bacillus subtilis Lacking Structural Maintenance of Chromosome Protein.

Bacillus subtilis mutants lacking the SMC-ScpAB complex are severely impaired for chromosome condensation and partitioning, DNA repair, and cells are not viable under standard laboratory conditions. We isolated suppressor mutations that restored the capacity of a smc deletion mutant (Δsmc) to grow under standard conditions. These suppressor mutations reduced chromosome segregation defects and abrogated hypersensitivity to gyrase inhibitors of Δsmc. Three suppressor mutations were mapped in genes involved in tRNA aminoacylation and maturation pathways. A transcriptomic survey of isolated suppressor mutations pointed to a potential link between suppression of Δsmc and induction of the stringent response. This link was confirmed by (p)ppGpp quantification which indicated a constitutive induction of the stringent response in multiple suppressor strains. Furthermore, sublethal concentrations of arginine hydroxamate (RHX), a potent inducer of stringent response, restored growth of Δsmc under non permissive conditions. We showed that production of (p)ppGpp alone was sufficient to suppress the thermosensitivity exhibited by the Δsmc mutant. Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor

The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome's central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA.

Functional interaction between ribosomal protein L6 and RbgA during ribosome assembly.

RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins.

Mutations in cell elongation genes mreB, mrdA and mrdB suppress the shape defect of RodZ‐deficient cells

RodZ interacts with MreB and both factors are required to maintain the rod shape of Escherichia coli. The assembly of MreB into filaments regulates the subcellular arrangement of a group of enzymes that synthesizes the peptidoglycan (PG) layer. However, it is still unknown how polymerization of MreB determines the rod shape of bacterial cells. Regulatory factor(s) are likely to be involved in controlling the function and dynamics of MreB. We isolated suppressor mutations to partially recover the rod shape in rodZ deletion mutants and found that some of the suppressor mutations occurred in mreB. All of the mreB mutations were in or in the vicinity of domain IA of MreB. Those mreB mutations changed the property of MreB filaments in vivo. In addition, suppressor mutations were found in the periplasmic regions in PBP2 and RodA, encoded by mrdA and mrdB genes. Similar to MreB and RodZ, PBP2 and RodA are pivotal to the cell wall elongation process. Thus, we found that mutations in domain IA of MreB and in the periplasmic domain of PBP2 and RodA can restore growth and rod shape to ΔrodZ cells, possibly by changing the requirements of MreB in the process.

Alternate Antioxidant Pathway

Biochemistry The low-molecular-weight thiol glutathione (GSH) is essential for growth in eukaryotes and plays a central role in stress responses and the maintenance of redox homeostasis in certain bacteria. Surprisingly, most aerobic bacteria lack the GSH biosynthesis enzyme glutamate cysteine ligase (GshA), although many have genes encoding proteins expected to require GSH for activity. To understand how these bacteria thrive, Veeravalli et al. isolated suppressor mutations in GshA-deficient Escherichia coli cells that expressed GSH. These cells carried mutations in the first two genes in the proline biosynthesis pathway, proB and proA . The mutations facilitated the accumulation of γ-glutamyl phosphate. This reacts with cysteine to give γ-glutamyl cysteine, which is converted to GSH by the enzyme glutathione synthase. Bioinformatic analysis revealed homologs of the suppressor ProB in bacterial strains that make GSH-dependent proteins but do not encode GshA. These results suggest that the proline biosynthetic pathway may have a role in GSH synthesis in prokaryotes. Nat. Chem. Biol. 7 , 101 (2011).

Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli

TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain.

Role of glutamate-126 and arginine-144 in the lactose permease of Escherichia coli.

Several previous studies have suggested that glutamate-126 and arginine-144 in the lactose permease of Escherichia coli form an ion pair that is essential for sugar binding. To further investigate the role of these residues, E126Q, R144Q, and R144S mutants were made. The R144Q and R144S strains, which had negligible levels of transport, were used as parental strains to isolate suppressor mutations that partially restored sugar transport. The R144Q parent only yielded first-site revertants, but the R144S strain produced three types of second-site replacements: E126Q, V229A, and L330R. In downhill transport assays, the E126Q strain was able to transport lactose at low levels, with an apparent K(m) 3-fold higher than the wild-type strain but a severely depressed apparent V(max). A triple mutant, E126Q/R144S/V229A, showed a relatively robust V(max) value for downhill transport and could actively accumulate lactose against a concentration gradient. Taken together, these results indicate that Glu-126 and Arg-144 are not essential for sugar binding. An alternative explanation for their role in maintaining secondary structure is discussed.

Mutations in sfdA and sfdB suppress multiple developmental mutations in Aspergillus nidulans.

Conidiophore morphogenesis in Aspergillus nidulans occurs in response to developmental signals that result in the activation of brlA, a well-characterized gene that encodes a transcription factor that is central to asexual development. Loss-of-function mutations in flbD and other fluffy loci have previously been shown to result in delayed development and reduced expression of brlA. flbD message is detectable during both hyphal growth and conidiation, and its gene product is similar to the Myb family of transcription factors. To further understand the regulatory pathway to brlA activation and conidiation, we isolated suppressor mutations that rescued development in strains with a flbD null allele. We describe here two new loci, designated sfdA and sfdB for suppressors of flbD, that bypass the requirement of flbD for development. sfd mutant alleles were found to restore developmental timing and brlA expression to strains with flbD deletions. In addition, sfd mutations suppress the developmental defects in strains harboring loss-of-function mutations in fluG, flbA, flbB, flbC, and flbE. All alleles of sfdA and sfdB that we have isolated are recessive to their wild-type alleles in diploids. Strains with mutant sfd alleles in otherwise developmentally wild-type backgrounds have reduced growth phenotypes and develop conidiophores in submerged cultures.

Hybrid protein between ribosomal protein S16 and RimM of Escherichia coli retains the ribosome maturation function of both proteins.

The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of E. coli approximately threefold. Several faster-growing derivatives of the rimM amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the rimM mRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the rimM mRNA. Three other independently isolated suppressor mutations created a fusion between rpsP, encoding the ribosomal protein S16, and rimM on the chromosome as a result of mutations in the rpsP stop codon preceding rimM. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.

The hgl1 gene is required for dimorphism and teliospore formation in the fungal pathogen Ustilago maydis.

The fungal pathogen Ustilago maydis causes a dramatic disease in maize involving the induction of tumours and the formation of masses of black teliospores. In this fungus, mating between haploid, budding cells results in the formation of the infectious, filamentous cell type that invades host tissue. Mating and filamentous growth are governed by the mating-type loci and by cAMP signalling, perhaps in response to signals from maize. To dissect the involvement of cAMP signalling further, the constitutive filamentous phenotype of a mutant with a defect in the catalytic subunit of protein kinase A was used to isolate suppressor mutations that restore budding growth. One such mutation identified the hgl1 gene, which is shown to be required for both the switch between budding and filamentous growth and teliospore formation during infection. In addition, the hgl1 gene product may be a target of phosphorylation by protein kinase A, and transcript levels for the gene are elevated during mating. Thus, the hgl1 gene provides a connection between mating, cAMP signalling and two important aspects of virulence: filamentous growth and the formation of teliospores.

Necrotic Neurodegeneration in Caenorhabditis Elegans.

Necrosis plays a central role in neuronal injury associated with stroke and ischemia. Unlike apoptotic cell death, little is understood of molecular mechanisms of necrosis. The two distinct forms of cell death, necrosis and apoptosis, are easily distinguishable by morphological characteristics in the nematode Caenorhabditis elegans. At the level of light microscopy, necrotic cells appear as swollen vacuoles several times larger than normal cells; apoptotic cells become compacted and raised. Under the electron microscope the first visible step in necroticlike death is the involution of the plasma membrane, followed by the appearance of membrane whorls [1]. Later, the electron density of the cytoplasm is reduced, the cell swells and cytoplasmic vacuoles arise. The chromatin forms aggregates and becomes fragmented. Organelles swell and finally lyse. The dead cell is removed by phagocytosis [2]. The key characterized initiators and regulators of apoptotic cell death do not influence the initiation or progression of necrotic cell death [2]. A variety of different insults can initiate necrotic-like neurodegeneration in C. elegans and lead to similar morphological changes in the affected cells. Necrosis-inducing genes include dominant alleles of the mec-4 and deg-1 degenerins which encode ion-channel subunits with similarity to mammalian epithelial Na + -ion channels (ENaC) [3, 4, 5], a dominant allele of the deg-3 acetylcholine receptor α-subunit [6], and a transgenically expressed constitutively activated mutant of the G-protein subunit GαS [7]. That channel hyperactivating mutations induce necrosis is reminiscent of the initiation of excitotoxic cell death in humans. In our lab we study necrotic neurodegeneration with a focus on channel-hyperactivating mutations in the mec-4 gene as the death-initiating event. The MEC-4 protein is expressed in the six touch neurons in C. elegans, where it participates in the formation of mechanically gated ion channels and is involved in the signal transduction of mechanical stimuli. The dominant mec-4(d) allele encodes a hyperactive channel that leads to necrotic demise of the touch receptor neurons. We established a behavioral assay that allowed us to isolate suppressor mutations that block mec-4(d)-induced neurodegeneration. Ectopic expression of mec-4(d) in the ventral nerve cord leads to the appearance of a number of swelling and degenerating cells visible as vacuoles during the larval stage L1. As a consequence of this cell death, worms are severely paralyzed. We mutagenized 45.000 haploid genomes and isolated 24 worms with reconstituted locomotion. Such strains harbor candidate suppressors of mec-4(d)-induced neurodegeneration. Ten of the suppressors proved to be alleles of mec-6, which is a known suppressor of ectopically expressed mec-4(d). The other loci define several previously unknown genes involved in mec-4(d)-induced cell death.

Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-[X]-R/K-R/K, of the lactose permease of Escherichia coli.

The lactose permease is a polytopic membrane protein that has a duplicated conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), located in cytoplasmic loops 2/3 and 8/9. In the current study, the roles of the basic residues and the acidic residue were investigated in greater detail. Neutral substitutions of two positive charges in loop 2/3 were tolerated, while a triple mutant resulted in a complete loss of expression. Neutral substitutions of a basic residue in loop 8/9 (i.e., K289I) also diminished protein stability. By comparison, neutral substitutions affecting the negative charge in loop 2/3 had normal levels of expression, but were defective in transport. A double mutant (D68T/N284D), in which the aspartate of loop 2/3 was moved to loop 8/9, did not have appreciable activity, indicating that the negative charge in the conserved motif could not be placed in loop 8/9 to recover lactose transport activity. An analysis of site-directed mutants in loop 7/8 and loop 8/9 indicated that an alteration in the charge distribution across transmembrane segment 8 was not sufficient to alleviate a defect caused by the loss of a negative charge in loop 2/3. To further explore this phenomenon, the double mutant, D68T/N284D, was used as a parental strain to isolate suppressor mutations which restored function. One mutant was obtained in which an acidic residue in loop 11/12 was changed to a basic residue (i.e., Glu374 --> Lys). Overall, the results of this study suggest that the basic residues in the conserved motif play a role in protein insertion and/or stability, and that the negative charge plays a role in conformational changes.

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal.

Subunit interactions in ABC transporters: aconserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits

The cytoplasmic membrane proteins of bacterial binding protein-dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA---G---------I-LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose-negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane-bound, as in wild-type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose-negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain.

Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

The AppA protein plays an essential regulatory role in development of the photosynthetic apparatus in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides 2.4.1 (M. Gomelsky and S. Kaplan, J. Bacteriol. 177:4609-4618, 1995). To gain additional insight into both the role and site of action of AppA in the regulatory network governing photosynthesis gene expression, we investigated the relationships between AppA and other known regulators of photosynthesis gene expression. We determined that AppA is dispensable for development of the photosynthetic apparatus in a ppsR null background, where PpsR is an aerobic repressor of genes involved in photopigment biosynthesis and puc operon expression. Moreover, all suppressors of an appA null mutation thus far isolated, showing improved photosynthetic growth, were found to contain mutations in the ppsR gene. Because ppsR gene expression in R. sphaeroides 2.4.1 appears to be largely independent of growth conditions, we suggest that regulation of repressor activity occurs predominately at the protein level. We have also found that PpsR functions as a repressor not only under aerobic but under anaerobic photosynthetic conditions and thereby is involved in regulating the abundance of the light harvesting complex II, depending on light intensity. It seems likely therefore, that PpsR responds to an integral signal (e.g., changes in redox potential) produced either by changes in oxygen tension or light intensity. The profile of the isolated suppressor mutations in PpsR is in accord with this proposition. We propose that AppA may be involved in a redox-dependent modulation of PpsR repressor activity.

Identification of global suppressors for temperature-sensitive folding mutations of the P22 tailspike protein

Suppressor mutations which alleviate the defects in folding mutants of the P22 gene 9 tailspike protein have recently been isolated (Fane, B. and King, J. (1991) Genetics 127, 263-277). The starting folding defects were in missense polypeptide chains generated by host amino acid insertions at different amber mutant sites. Fragments of genes carrying the amber mutations with and without their independently isolated suppressor mutations were cloned and sequenced. The parental nonsense mutations were located at Q45, K122, E156, W202, W207, Y232, and W365. Their conformational suppressors were single amino acid substitutions at a limited set of sites, V84 greater than A, V331 greater than A, and A334 greater than V. The V331 greater than A or A334 greater than V suppressors were independently recovered starting with different mutant sites suggesting that they acted by some global or general mechanism. When the V331 greater than A and A334 greater than V mutations were crossed into well-characterized temperature-sensitive folding (tsf) mutants at various sites in the tailspike protein, they suppressed all of the eight tsf mutants tested. Since the tsf defects destabilize folding intermediates rather than the native conformation, this result implies that the suppressors act in the folding pathway. Strains carrying the isolated suppressor mutations displayed no obvious phenotypic defect and formed native biologically active tailspikes. Thus, these single amino acid substitutions have striking influences on the efficiency of intracellular chain folding, without causing functional defects in the native protein.