A family of modular type mannuronan C-5-epimerase genes controls alginate structure in Azotobacter vinelandii

The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR-PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), β-lactamase (monomeric, ToxR–Bla), or the leucine zipper of GCN4 (dimeric, ToxR-GCN4-M) could substitute functionally for the ToxR periplasmic domain. ToxR-GCN4 fusion proteins, in which the ToxR trans-membrane domain was eliminated (ToxR-GCN4-C), were inactive, but an additional fusion protein that contained a heterologous membrane-spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR-GCN4-M and ToxR-Bla responded like wild-type strains to in vitro growth conditions. In the infant-mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild-type ToxR, and strains containing monomeric ToxR-Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.     

霍乱弧菌ToxS蛋白间的互作增强ToxR蛋白诱导毒力基因的表达

【目的】阐明霍乱弧菌ToxR蛋白功能调控的分子机制。【方法】利用巯基捕获(thiol-trapping)的方法分析DsbA蛋白对ToxR周质空间结构域半胱氨酸残基的氧化作用;采用定点突变的方法构建ToxR半胱氨酸突变株(ToxR_(C236/293S));利用荧光素酶基因作为报告基因分析ToxR野生型(ToxR_(wt))和半胱氨酸突变体(ToxR_(C236/293S))诱导下游基因表达的活性;通过细菌双杂交系统分析ToxR_(wt)和ToxR_(C236/293S)蛋白之间、ToxR与ToxS之间以及ToxS之间的相互作用。【结果】ToxR周质空间结构域半胱氨酸残基确实可以被DsbA蛋白氧化,且当ToxR与ToxS共表达时,ToxR诱导ctxAB转录表达的活性显著增强,且在dsbA基因缺失突变株中ToxR诱导ctxAB转录表达的活性更高;成功构建株霍乱弧菌ToxR半胱氨酸突变株(ToxR_(C236/293S)),在没有ToxS存在的条件下,ToxR_(C236/293S)诱导毒力基因表达的活性与ToxRwt相当;细菌双杂交系统分析发现当ToxR与ToxS共转录表达时,ToxS极大增强ToxR蛋白之间的互作;在dsbA基因缺失突变株中,ToxS之间的相互作用显著增强。【结论】ToxR蛋白本身的氧还状态对其诱导毒力基因表达的活性没有影响;ToxS通过增强ToxR形成二聚体的能力从而增强其诱导毒力基因的表达,而DsbA对ToxS蛋白之间的相互作用具有抑制作用,DsbA通过影响ToxS的蛋白互作从而影响ToxR蛋白的功能。本文为进一步阐明霍乱弧菌毒力基因表达调控的分子机制提供重要的理论依据。     

Analysis of membrane protein interaction: ToxR can dimerize the amino terminus of phage lambda repressor

The ToxR protein of Vibrio cholerae is an integral membrane protein that co-ordinately regulates virulence determinant expression. ToxR directiy activates the cholera toxin operon, but maximal activation is achieved in the presence of ToxS, an integral membrane protein thought to interact with ToxR periplasmic sequences. Studies that substitute alkaline phosphatase sequences for the periplasmic domain of ToxR have led to a model for ToxR activation based on dimerization and ToxS interaction. We constructed λ-ToxR chimeric proteins using the DNA-binding domain of the phage λ repressor, which cannot effectively dimerize by itself, to assess the ability of ToxR to form dimers in Escherichia coli The results suggest that ToxR sequences can propagate dimerization, and that ToxS can influence the ability to dimerize.     

A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation

ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.     

Role of ToxS in the proteolytic cascade of virulence regulator ToxR in Vibrio cholerae

Two of the primary virulence regulators of Vibrio cholerae, ToxR and TcpP, function together with cognate effector proteins. ToxR undergoes regulated intramembrane proteolysis (RIP) during late stationary phase in response to nutrient limitation at alkaline pH; however, the specific function of its cognate ToxS remains unresolved. In this work, we found that ToxR rapidly becomes undetectable in a ΔtoxS mutant when cultures are exposed to either starvation conditions or after alkaline pH shock individually. A ΔtoxS mutant enters into a dormant state associated with the proteolysis of ToxR at a faster rate than wild‐type, closely resembling a ΔtoxR mutant. Using a mutant with a periplasmic substitution in ToxS, we found that the proteases DegS and DegP function additively with VesC and a novel protease, TapA, to degrade ToxR in the mutant. Overall, the results shown here reveal a role for ToxS in the stabilization of ToxR by protecting the virulence regulator from premature proteolysis.     

A Two-Plasmid System for Independent Genetic Manipulation of Subunits of Homodimeric Proteins and Selective Isolation of Chimeric Dimers

We have designed and tested a modular two-plasmid expression system which allows coexpression of two different subunits of recombinant dimeric protein in Escherichia coli and selective purification of heterodimers. We have constructed a new expression vector, pBIOEx, with p15a replication origin which allows its stable coexistence with different ColE1 group plasmids. The expression cassette of this plasmid under control of the T7 promoter contains cloning site, followed by a short sequence coding for the C-terminal extension of the recombinant protein which is a target of the in vivo biotinylation by BirA protein. The expression unit is bicistronic, the second expressed protein being BirA. We have used this plasmid together with pET30a to clone kinesin heavy-chain fragment and coexpressed the two polypeptide chains differing by tags on their C-termini and we purified heterodimers made of two recombinant molecules. The heterodimeric protein had a normal biochemical activity. There was no discrimination against heterodimer formation at the dimerization step. The system is a powerful tool in studies of different aspects of interactions between subunits of the homodimeric proteins since it makes possible separate genetic manipulations on each subunit of the dimer.     

Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS(Vc)) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS(Vp)), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR(Vv), toxS(Vv), and htpG(Vv) were cloned. ToxR(Vv) shared 55.0 and 63.0% sequence homology with ToxR(Vc) and ToxR(Vp), respectively. ToxS(Vv) was 71.5 and 65.7% homologous to ToxS(Vc) and ToxS(Vp), respectively. The amino acid sequences of ToxRS(Vv) showed transmembrane and activity domains similar to those observed in ToxRS(Vc) and ToxRS(Vp). Western blot analysis proved the expression of ToxR(Vv) in V. vulnificus. ToxRS(Vv) enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRS(Vv) also activated the ToxR(Vc)-regulated ctx promoter incorporated into an E. coli chromosome. A toxR(Vv) null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR(Vv) mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR(Vv) may regulate the virulence expression of V. vulnificus.     

Biophysical studies of the membrane-embedded and cytoplasmic forms of the glucose-specific Enzyme II of the E. coli phosphotransferase system (PTS)

The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-II(Glc)-YFP and MBP-II(Glc)-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET(-)) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET(+)). The monomeric species could form a heterodimeric species (FRET(+)) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-II(Glc) activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-II(Glc) retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-II(Glc) indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-II(Glc).     

The stability and formation of native proteins from unfolded monomers is increased through interactions with unrelated proteins

The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.     

A molecular dynamics study of the formation, stability, and oligomerization state of two designed coiled coils: possibilities and limitations

The formation, relative stability, and possible stoichiometries of two (self-)complementary peptide sequences (B and E) designed to form either a parallel homodimeric (B + B) or an antiparallel heterodimeric (B + E) coiled coil have been investigated. Peptide B shows a characteristic coiled coil pattern in circular dichroism spectra at pH 7.4, whereas peptide E is apparently random coiled under these conditions. The peptides are complementary to each other, with peptide E forming a coiled coil when mixed with peptide B. Molecular dynamics simulations show that combinations of B + B and B + E readily form a dimeric coiled coil, whereas E + E does not fall in line with the experimental data. However, the simulations strongly suggest the preferred orientation of the helices in the homodimeric coiled coil is antiparallel, with interactions at the interface quite different to that of the idealized model. In addition, molecular dynamics simulations suggest equilibrium between dimers, trimers, and tetramers of alpha-helices for peptide B.