A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif

The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.     

Twin‐arginine translocation system (tat) mutants of Salmonella are attenuated due to envelope defects,not respiratory defects

The twin‐arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat‐exported protein and determined the virulence phenotype of mutant strains. Although a tat mutant is highly attenuated, no single Tat‐exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N‐acetylmuramoyl‐l ‐alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments.     

Role of the Escherichia coli Tat pathway in outer membrane integrity

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.     

Characterization of the Twin-Arginine Transport Secretome in Sinorhizobium meliloti and Evidence for Host-Dependent Phenotypes

The twin-arginine transport (Tat) system is essential for cell viability in Sinorhizobium meliloti and may play a role during the development of root nodules. Utilizing an in vivo recombination strategy, we have constructed 28 strains that contain deletions in predicted Tat substrates. Testing of these mutations for symbiotic proficiency on the plant hosts alfalfa and sweet clover shows that some of these mutations affect associations with these hosts differentially.     

Trans-dominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is required for efficient viral gene expression. By means of mutational analyses, several domains of the Tat protein that are required for complete activation of HIV-1 gene expression have been defined. These include an amino-terminal activating domain, a cysteine-rich dimerization domain, and a basic domain important in the binding of Tat to the trans-activation response element (TAR) and in Tat nuclear localization. Recently, we described a mutation, known as delta tat, which resulted in a protein with a truncated basic domain. This protein had a "trans-dominant" phenotype in that it inhibited wild-type Tat activation of the HIV-1 LTR. To further characterize the requirements for generating a Tat trans-dominant phenotype, we constructed a variety of Tat proteins with truncations or substitutions in the basic domain. A number of these proteins showed a trans-dominant phenotype. These Tat mutants also inhibited activation of the HIV-1 LTR by a protein composed of Tat fused to the prokaryotic R17 (phage MS2) RNA-binding protein in which the R17 recognition element was inserted in the HIV-1 LTR in place of TAR. Thus, an intact TAR element was not required for this inhibition. We also studied the cellular localization of Tat and a trans-dominant Tat mutant by means of immunofluorescence staining with the use of antibodies reactive to different domains of the Tat protein. The results indicated that Tat becomes localized predominantly in the nucleus both in the presence and absence of the trans-dominant Tat construct, suggesting that the trans-dominant mutant does not inhibit Tat nuclear localization. These studies further define the requirements for the creation of trans-dominant Tat mutants, and suggest that the mechanism of trans-dominant Tat inhibition may be either the formation of an inactive complex between wild-type and mutant Tat or sequestration of cellular factors involved in regulating HIV-1 gene expression.     

The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> under high-pressure and low-temperature conditions

Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2^E27F) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2^D563N) and HPG2-3 (Tat2^E570K) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.Communicated by K. Horikoshi     

Hyper-mutation caused by the reml mutation in yeast is not dependent on error-prone or excision repair

The reml mutations of Saccharomyces cerevisiae confer a semi-dominant hyper-recombination/hyper-mutation phenotype. Neither reml mutant allele has any apparent meiotic affect. We have examined spontaneous mutation in reml-2 strains and demonstrate that the reml-2 mutation, like reml-1, confers an average 10-fold increase in reversion and forward mutation rates. Unlike certain yeast rad mutations with phenotypes similar to reml, strains containing reml are resistant to MMS and only slightly UV sensitive at very high doses. To understand the mutator phenotype of reml, we have used a double-mutant approach, combining the reml mutation with radiation-sensitive mutations affecting DNA repair. Double mutants of reml-2 and a mutation in the yeast error-prone repair group (rad6-1) or a mutation in excision repair (rad1-2 or rad4) maintain the hyper-mutation phenotype. Since mutation rates remain elevated in these double-mutant strains, it appears as if the mutations which occur in the presence of reml resemble spontaneous mutation since they do not require the action of a repair system.     

Inhibition of human immunodeficiency virus type 1 and type 2 Tat function by transdominant Tat protein localized to both the nucleus and cytoplasm.

We introduced various mutations into the activation and RNA binding domains of human immunodeficiency virus type 1 (HIV-1) Tat in order to develop a novel and potent transdominant Tat protein and to characterize its mechanism of action. The different mutant Tat proteins were characterized for their abilities to activate the HIV LTR and inhibit the function of wild-type Tat in trans. A Tat protein containing a deletion of the basic domain (Tat(delta)49-57) localized exclusively to the cytoplasm of transfected human cells was nonfunctional and inhibited both HIV-1 and HIV-2 Tat function in a transdominant manner. Tat proteins containing mutations in the cysteine-rich and core domains were nonfunctional but failed to inhibit Tat function in trans. When Tat nuclear or nucleolar localization signals were fused to the carboxy terminus of Tat(delta)49-57, the chimeric proteins localized to the nucleus or nucleolus, respectively, and remained capable of acting in a transdominant manner. Introduction of secondary mutations in the cysteine-rich and core domains of the various transdominant Tat proteins completely eliminated their abilities to act in a transdominant fashion. Our data best support a mechanism in which these transdominant Tat proteins squelch a cellular factor or factors that interact with the Tat activation domain and are required for Tat to function.     

Mutations in nif genes that cause Klebsiella pneumoniae to be derepressed for nitrogenase synthesis in the presence of ammonium.

Four Nif+ revertants from strains with polar insertions in nifL, were insensitive to ammonium and amino acid repression of nitrogenase synthesis. These strains have mutations located in or near the nifL region. The derepressed phenotype was dominant in a merodiploid containing a nif+ plasmid. These nif regulatory mutations also suppressed the Nif- phenotype of Gln- strains. Thus, regulation by fixed nitrogen (possible via glutamine synthetase) occurs on the nifLA operon but not on the other six nif operons.     

Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin-arginine translocase

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter-subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions. We show that substitution in TatB of three residues by glycine, or a single residue by proline, has no detectable effect on translocation, whereas the presence of three glycines in the TatA TM span completely blocks Tat translocation activity. The results show that the integrity of the TatA TM span is vital for Tat activity, whereas that of TatB can accommodate large-scale distortions. Near-complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction between the TatA/B TM spans. We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction; benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination. Finally, we have re-examined the effects of previously analyzed TatABC mutations under conditions of high translocation activity. Among numerous TatA or TatB mutations tested, TatA F39A alone blocked translocation, and only substitutions of P48 and F94 in TatC blocked translocation activity.     

Analysis of conditional mutations in the Saccharomyces cerevisiae MLH1 gene in mismatch repair and in meiotic crossing over

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.     

Genes other than TLR4 are involved in the response to inhaled LPS

For several decades, the mouse strains C3H/HeJ and C57BL/10ScNCr have been known to be hyporesponsive to endotoxin or lipopolysaccharide (LPS). Recently, mutations in Toll-like receptor (TLR) 4 have been shown to underlie this aberrant response to LPS. To further determine the relationship between TLR4 and responsiveness to LPS, we genotyped 18 strains of mice for TLR4 and evaluated the physiological and biological responses of these strains to inhaled LPS. Of the 18 strains tested, 6 were wild type for TLR4 and 12 had mutations in TLR4. Of those strains with TLR4 mutations, nine had mutations in highly conserved residues. Among the strains wild type for TLR4, the inflammatory response in the airway induced by inhalation of LPS showed a phenotype ranging from very sensitive (DBA/2) to hyporesponsive (C57BL/6). A broad spectrum of airway hyperreactivity after inhalation of LPS was also observed among strains wild type for TLR4. Although the TLR4 mutant strains C3H/HeJ and C57BL/10ScNCr were phenotypically distinct from the other strains with mutations in the TLR4 gene, the other strains with mutations for TLR4 demonstrated a broad distribution in their physiological and biological responses to inhaled LPS. The results of our study indicate that although certain TLR4 mutations can be linked to a change in the LPS response phenotype, additional genes are clearly involved in determining the physiological and biological responses to inhaled LPS in mammals.     

Localization of the Tat translocon components in Escherichia coli

The Tat system has the ability to translocate folded proteins across the bacterial cytoplasmic membrane. In Escherichia coli, three functionally different translocon components have been identified, namely TatA, TatB, and TatC. These proteins were fused to the green fluorescent protein (GFP) and their localization was determined by confocal laser scanning fluorescence microscopy. TatA-GFP was distributed in the membrane, often with higher abundance at the poles. TatB-GFP was found in distinct spots at the poles of the cells. The fluorescence of TatC-GFP was very low and required a constitutive expression system to become higher than background, but then appearing polar. All three constructs complemented the chain-formation phenotype of corresponding mutant strains, indicating the functionality of the fusion proteins. TatB-GFP and TatC-GFP also complemented TMAO respiration deficiency and TatA-GFP the SDS-sensitivity of the mutant strains. The localization of the translocon-GFP fusions coincides with the fluorescence pattern of GFP fusions to Tat substrate signal sequences. We suggest that the active translocon complexes are mainly present at polar positions in Escherichia coli.     

Phenotypic switching in Cryptococcus neoformans

Cryptococcus neoformans strains exhibit considerable phenotype variability with regards to the capsular polysaccharide, sterol composition of the cell wall, and cell and colony morphology. Phenotypic changes can occur spontaneously during in vitro passage of strains or during chronic infection in vivo and may be associated with differences in virulence. Studies from our laboratory have demonstrated that phenotype variability can be the result of phenotypic switching. Phenotypic switching is defined as a reversible change of an observable colony phenotype that occurs at a frequency above the expected frequency for somatic mutations. This implies that phenotypic switching represents controlled and programmed changes in this pathogenic yeast rather than random mutations. We have shown that a phenotypic switch from a smooth colony phenotype to a mucoid colony phenotype occurs in vitro and in vivo during chronic infection of mice. More importantly we have now demonstrated that the switch is associated with an increase in virulence and a change in the host immune response. Implications of these findings for the pathogenesis of cryptococcosis are discussed.     

Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF

We recently demonstrated that Pseudomonas aeruginosa PAO1 undergoes a pronounced phenotypic change when introduced into the intestines of rats during surgical injury. Recovered strains displayed a specific phenotype (termed the P2 phenotype) characterized by altered pyocyanin production, high collagenase activity, high swarming motility, low resistance to chloramphenicol, and increased killing of Caenorhabditis elegans compared to the inoculating strain (termed the P1 phenotype). The aims of this study were to characterize the differences between the P. aeruginosa P1 and P2 phenotypes in quorum sensing and competitiveness. We then determined the presence of the P2 phenotype among PAO1 strains from various laboratories. Results demonstrated that P2 cells display accelerated growth during early exponential phase and early activation of quorum-sensing systems and overcome the growth of P1 cells in a mixed population. Among eight PAO1 strains obtained from different laboratories, four exhibited the P2 phenotype. Of 27 mutants analyzed from the P. aeruginosa MPAO1 transposon library, 25 displayed P2 phenotypes. The P2 phenotype in both cases correlated with a lack of expression of mexE or mexF due to mutations in mexT and mexF genes. In summary, strains possessing the P2 phenotype are distributed among PAO1 strains commonly used across a variety of research laboratories. Genetically, they are characterized by various mutations in mexT or mexF.     

Expression level of heterologous tat genes is crucial for in vivo reconstitution of a functional Tat translocase in Escherichia coli

The Tat system has the remarkable capacity of exporting proteins in folded conformation across the cytoplasmic membrane. The functional Tat translocase from Gram-negative bacteria consists of TatA, TatB and TatC proteins. To gain information about the species specificity of the Tat translocase, we cloned tat genes from Gram-negative pathogens Shigella flexneri 2a str. 301, Vibrio cholerae El Tor N16961, Pseudomonas aeruginosa PAO1, thermophilic Sulfolobus solfataricus P2, Thermus thermophilus HB8 and from three Magnetospirillum species (AMB-1, MS-1 and MSR-1), and assessed the capacity of these Tat systems to restore the Tat-dependent growth defect of Escherichia coli tat mutants. We found that whereas the tat genes from the thermophilic bacterial and archaeal species were not functional in E. coli, other tat genes could all complement the phenotype of the E. coli tat mutants. In addition, a chimera composed of the N-terminus of V. cholerae TatE and C-terminus of M. magneticum TatA was functional. Whereas the expression of the tatABC genes from P. aeruginosa and Magnetospirillum strains must be induced to obtain a functional Tat system, overproduction of the V. cholerae TatABC proteins abolished the complementation. The complementation impairment seemed to be correlated with increasing level of slow-migrating TatC isoforms. In vitro studies showed that slow-migrating TatC isoforms in the purified V. cholerae TatABC complex increased with storage time. Together these results showed that the Tat translocases from the Gram-negative bacteria are generally functional in E. coli and the expression level is crucial for in vivo reconstitution of a functional Tat translocase.     

Full peptide synthesis, purification, and characterization of six Tat variants. Differences observed between HIV-1 isolates from Africa and other continents

AIDS in Africa is characterized by the equal distribution of mortality between the two genders because of highly virulent human immunodeficiency virus type 1 (HIV-1) strains. The viral protein Tat trans-activates viral gene expression and is essential for HIV-1 replication. We chemically synthesized six different Tat proteins, with sizes ranging from 86 to 101 residues, from HIV-1 isolates located in different parts of the world including highly virulent African strains. Protein purification, mass spectroscopy, and amino acid analysis showed that the synthesis was successful in each case but with different yields. We show that all have the ability to bind the HIV long terminal repeat (LTR) RNA trans-activation response element (TAR) region, involved in Tat-mediated trans-activation, but structural heterogeneities are revealed by circular dichroism. These Tat synthetic proteins cross membranes but differ in their ability to trans-activate an HIV LTR-reporter gene in stably transfected HeLa cells. Two Tat proteins from virulent African HIV-1 strains were much more active than those from Europe and the United States. The interferon-induced kinase (PKR), involved in cell antiviral defense, phosphorylates only Tat variants corresponding to less or nonvirulent HIV-1 isolates. Our results indicate that the high virulence of some African HIV-1 strains could be related to Tat activity.