The Long Hunt for pssR-Looking for a Phospholipid Synthesis Transcriptional Regulator, Finding the Ribosome.

The phospholipid (PL) composition of bacterial membranes varies as a function of growth rate and in response to changes in the environment. While growth adaptation can be explained by biochemical feedback in the PL synthesis pathway, recent transcriptome studies have revealed that the expression of PL synthesis genes can also be tuned in response to various stresses. We previously showed that the BasRS two-component pathway controls the expression of the diacylglycerol kinase gene, dgkA, in Escherichia coli (A. Wahl, L. My, R. Dumoulin, J. N. Sturgis, and E. Bouveret, Mol Microbiol, 80:1260-1275, 2011, https://doi.org/10.1111/j.1365-2958.2011.07641.x). In this study, we set up a strategy to identify the mutation responsible for the upregulation of pssA observed in the historical pssR1 mutant and supposedly corresponding to a transcriptional repressor (C. P. Sparrow and J. Raetz, J Biol Chem, 258:9963-9967, 1983). pssA encodes phosphatidylserine synthase, the first step of phosphatidylethanolamine synthesis. We showed that this mutation corresponded to a single nucleotide change in the anti-Shine-Dalgarno sequence of the 16S rRNA encoded by the rrnC operon. We further demonstrated that this mutation enhanced the translation of pssA Though this effect appeared to be restricted to PssA among phospholipid synthesis enzymes, it was not specific, as evidenced by a global effect on the production of unrelated proteins.IMPORTANCE Bacteria adjust the phospholipid composition of their membranes to the changing environment. In addition to enzymatic regulation, stress response regulators control specific steps of the phospholipid synthesis pathway. We wanted to identify a potential regulator controlling the expression of the phosphatidylserine synthase gene. We showed that it was not the previously suggested hdfR gene and instead that a mutation in the anti-Shine-Dalgarno sequence of 16S RNA was responsible for an increase in pssA translation. This example underlines the fact that gene expression can be modulated by means other than specific regulatory processes.

Reassessment of the Genetic Regulation of Fatty Acid Synthesis in Escherichia coli: Global Positive Control by the Dual Functional Regulator FadR.

UNLABELLED: In Escherichia coli, the FadR transcriptional regulator represses the expression of fatty acid degradation (fad) genes. However, FadR is also an activator of the expression of fabA and fabB, two genes involved in unsaturated fatty acid synthesis. Therefore, FadR plays an important role in maintaining the balance between saturated and unsaturated fatty acids in the membrane. We recently showed that FadR also activates the promoter upstream of the fabH gene (L. My, B. Rekoske, J. J. Lemke, J. P. Viala, R. L. Gourse, and E. Bouveret, J Bacteriol 195:3784-3795, 2013, doi:10.1128/JB.00384-13). Furthermore, recent transcriptomic and proteomic data suggested that FadR activates the majority of fatty acid (FA) synthesis genes. In the present study, we tested the role of FadR in the expression of all genes involved in FA synthesis. We found that FadR activates the transcription of all tested FA synthesis genes, and we identified the FadR binding site for each of these genes. This necessitated the reassessment of the transcription start sites for accA and accB genes described previously, and we provide evidence for the presence of multiple promoters driving the expression of these genes. We showed further that regulation by FadR impacts the amount of FA synthesis enzymes in the cell. Our results show that FadR is a global regulator of FA metabolism in E. coli, acting both as a repressor of catabolism and an activator of anabolism, two directly opposing pathways. IMPORTANCE: In most bacteria, a transcriptional regulator tunes the level of FA synthesis enzymes. Oddly, such a global regulator still was missing for E. coli, which nonetheless is one of the prominent model bacteria used for engineering biofuel production using the FA synthesis pathway. Our work identifies the FadR functional dual regulator as a global activator of almost all FA synthesis genes in E. coli. Because FadR also is the repressor of FA degradation, FadR acts both as a repressor and an activator of the two opposite pathways of FA degradation and synthesis. Our results show that there are still discoveries waiting to be made in the understanding of the genetic regulation of FA synthesis, even in the very well-known bacterium E. coli.

New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification.

We report the first use of tandem affinity purification (TAP) in a prokaryote to purify native protein complexes, and demonstrate its reliability and power. We purified the acyl carrier protein (ACP) of Escherichia coli, a protein involved in a myriad of metabolic pathways. Besides the identification of several known partners of ACP, we rediscovered ACP/MukB and ACP/IscS interactions already detected but previously disregarded as due to contamination. Here, we demonstrate the specificity of these interactions and characterize them. This suggests that ACP is involved in additional previously unsuspected pathways. Furthermore, this study shows how the TAP method can be simply used in prokaryotes such as E. coli to identify new partners in protein-protein interactions under physiological conditions and thereby uncover novel protein functions.

Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm.

Several proteins of the Tol/Pal system are required for group A colicin import into Escherichia coli. Colicin A interacts with TolA and TolB via distinct regions of its N-terminal domain. Both interactions are required for colicin translocation. Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR. This interaction is specific to colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the colicin translocation step. TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm. The interaction involves part of the periplasmic domain of TolR and a small region of the colicin A N-terminal domain. This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the colicin A N-terminal domain and appear to be arranged linearly in the colicin sequence. Multiple contacts with periplasmic-exposed Tol proteins are therefore a general principle required for group A colicin translocation.

The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?

The outer membrane of gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities.

The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.

Identification of phospholipids as new components that assist in the in vitro trimerization of a bacterial pore protein.

The in vitro trimerization of folded monomers of the bacterial pore protein PhoE, into its native-like, heat- and SDS-stable form requires incubations with isolated cell envelopes and Triton X-100. The possibility that membranes could be isolated that are enriched in assembly factors required for assembly of the pore protein was now investigated. Fractionation of total cell envelopes of Escherichia coli via various techniques indeed revealed the existence of membrane fractions with different capacities to support assembly in vitro. Fractions containing mainly inner membrane vesicles supported the formation of trimers that were associated with these membrane vesicles. However, only a proportion of these trimers were heat- and SDS-stable and these were formed with slow kinetics. In contrast, fractions containing mainly outer membrane vesicles supported formation of high amounts of heat-stable trimers with fast kinetics. We identified phospholipids as active assembly components in these membranes that support trimerization of folded monomers in a process with similar characteristics as observed with inner membrane vesicles. Furthermore, phospholipids strongly stimulate the kinetics of trimerization and increase the final yield of heat-stable trimers in the context of outer membranes. We propose that lipopolysaccharides stabilize the assembly competent state of folded monomers as a lipochaperone. Phospholipids are involved in converting the folded monomer into new assembly competent intermediate with a short half-life that will form heat-stable trimers most efficiently in the context of outer membrane vesicles. These results provide biochemical evidence for the involvement of different lipidic components at distinct stages of the porin assembly process.

A Sm-like protein complex that participates in mRNA degradation.

In eukaryotes, seven Sm proteins bind to the U1, U2, U4 and U5 spliceosomal snRNAs while seven Smlike proteins (Lsm2p-Lsm8p) are associated with U6 snRNA. Another yeast Sm-like protein, Lsm1p, does not interact with U6 snRNA. Surprisingly, using the tandem affinity purification (TAP) method, we identified Lsm1p among the subunits associated with Lsm3p. Coprecipitation experiments demonstrated that Lsm1p, together with Lsm2p-Lsm7p, forms a new seven-subunit complex. We purified the two related Sm-like protein complexes and identified the proteins recovered in the purified preparations by mass spectrometry. This confirmed the association of the Lsm2p-Lsm8p complex with U6 snRNA. In contrast, the Lsm1p-Lsm7p complex is associated with Pat1p and Xrn1p exoribonuclease, suggesting a role in mRNA degradation. Deletions of LSM1, 6, 7 and PAT1 genes increased the half-life of reporter mRNAs. Interestingly, accumulating mRNAs were capped, suggesting a block in mRNA decay at the decapping step. These results indicate the involvement of a new conserved Sm-like protein complex and a new factor, Pat1p, in mRNA degradation and suggest a physical connection between decapping and exonuclease trimming.

Structure of the Escherichia coli TolB protein determined by MAD methods at 1.95 A resolution.

BACKGROUND: The periplasmic protein TolB from Escherichia coli is part of the Tol-PAL (peptidoglycan-associated lipoprotein) multiprotein complex used by group A colicins to penetrate and kill cells. TolB homologues are found in many gram-negative bacteria and the Tol-PAL system is thought to play a role in bacterial envelope integrity. TolB is required for lethal infection by Salmonella typhimurium in mice. RESULTS: The crystal structure of the selenomethionine-substituted TolB protein from E. coli was solved using multiwavelength anomalous dispersion methods and refined to 1. 95 A. TolB has a two-domain structure. The N-terminal domain consists of two alpha helices, a five-stranded beta-sheet floor and a long loop at the back of this floor. The C-terminal domain is a six-bladed beta propeller. The small, possibly mobile, contact area (430 A(2)) between the two domains involves residues from the two helices and the first and sixth blades of the beta propeller. All available genomic sequences were used to identify new TolB homologues in gram-negative bacteria. The TolB structure was then interpreted using the observed conservation pattern. CONCLUSIONS: The TolB beta-propeller C-terminal domain exhibits sequence similarities to numerous members of the prolyl oligopeptidase family and, to a lesser extent, to class B metallo-beta-lactamases. The alpha/beta N-terminal domain shares a structural similarity with the C-terminal domain of transfer RNA ligases. We suggest that the TolB protein might be part of a multiprotein complex involved in the recycling of peptidoglycan or in its covalent linking with lipoproteins.

In vitro characterization of peptidoglycan-associated lipoprotein (PAL)-peptidoglycan and PAL-TolB interactions.

The Tol-peptidoglycan-associated lipoprotein (PAL) system of Escherichia coli is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan. Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan. We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation. Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan. A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan. Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive. Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan. This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan.

Crystallization and preliminary crystallographic study of a component of the Escherichia coli tol system: TolB.

TolB from Escherichia coli is part of the Tol system used by the group A colicins to penetrate and kill cells. A TolB derivative tagged with six histidines was overexpressed, purified by chelation on a nickel affinity column and crystallized using the SAmBA software to define the optimal crystallization protocol. The crystals belong to the monoclinic system, space group P21 with unit-cell parameters a = 64.48, b = 41.06, c = 78.41 A, beta = 110.78 degrees. Frozen crystals diffract to 1.9 A resolution. Screening for heavy-atom derivatives both on the native TolB and various cysteine-substituted mutants is in progress. In addition, a selenomethionine-substituted protein is being produced in order to use the MAD method for structure determination.

Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli.

Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N-terminal domain of colicins is involved in the import process. The N-terminal domains of colicins A and E1 have been shown to interact with TolA, and the N-terminal domain of colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N-terminal domain of all group A colicins, the 'TolA box', was important for colicin A import but was not involved in the colicin A-TolA interaction. It was, however, involved in the colicin A-TolB interaction. The interactions of colicin A N-terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the colicin A N-terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild-type domain of the entire colicin A for import into the cells. Our results demonstrate that different regions of the colicin A N-terminal domain interact with TolA and TolB. The colicin A N-terminal domain was also shown to form a trimeric complex with TolA and TolB.

The TolB protein interacts with the porins of Escherichia coli.

TolB is a periplasmic protein of the cell envelope Tol complex. It is partially membrane associated through an interaction with the outer membrane lipoprotein PAL (peptidoglycan-associated lipoprotein), which also belongs to the Tol system. The interaction of TolB with outer membrane porins of Escherichia coli was investigated with a purified TolB derivative harboring a six-histidine tag. TolB interacted with the trimeric porins OmpF, OmpC, PhoE, and LamB but not with their denatured monomeric forms or OmpA. These interactions took place both in the presence and in the absence of lipopolysaccharide. TolA, an inner membrane component of the Tol system, also interacts with the trimeric porins via its central periplasmic domain (R. Dérouiche, M. Gavioli, H. Bénédetti, A. Prilipov, C. Lazdunski, and R. Lloubès, EMBO J. 15:6408-6415, 1996). In the presence of the purified central domain of TolA (TolAIIHis), the TolB-porin complexes disappeared to form TolAIIHis-porin complexes. These results suggest that the interactions of TolA and TolB with porins might take place in vivo and might be concomitant events participating in porin assembly. They also suggest that the Tol system as a whole may be involved in porin assembly in the outer membrane.

The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step.

Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli: the Tol system for group A colicins and the TonB system for group B colicins. The N-terminal domain of colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N-terminal domains of colicin E3 (group A) and colicin B (group B). Producing cells became specifically tolerant to entire extracellular colicins of the same group. The periplasmic N-terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross-linking and co-immunoprecipitation experiments in cells producing the colicin E3 N-terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB. After in vitro cross-linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain. The N-terminal domain of a translocation-defective colicin E3 mutant was found to no longer interact with TolB. Hence, this interaction must play an important role in colicin E3 translocation.

Peptidoglycan-associated lipoprotein-TolB interaction. A possible key to explaining the formation of contact sites between the inner and outer membranes of Escherichia coli.

TolA, -B, -Q, and -R proteins are involved in maintaining the cell envelope integrity of Escherichia coli; they have been parasitized by the group A colicins and the single strand DNA of some filamentous bacteriophages to permit them to enter the cells. TolA and TolR are anchored to the inner membrane by a single transmembrane domain, TolQ is an integral membrane protein with three transmembrane segments, and TolB has recently been found to be periplasmic although it is partially membrane-associated. The latter result suggests that TolB might interact with membrane proteins. Other lines of evidence favor the existence of a Tol complex. To further characterize this complex, we investigated which proteins interact with TolB. For this purpose, two different methods were used. First, we took advantage of the existence of a tagged TolB (TolBep) to perform immunoprecipitation under native conditions in order to preserve the putative associations of TolBep with other proteins. Secondly, in vivo cross-linking experiments with formaldehyde were performed. These two approaches led to the same result and demonstrated for the first time that a component of the Tol system, TolB, interacts with a protein located in the outer membrane, the peptidoglycan-associated lipoprotein.

[Curative activity of spiramycin adipate by parenteral route in experimental septicemia in mice. Comparison with orally administered basic spiramycin]. / Activité curative de l'adipate de spiramycine utilisé par voie parentérale dans la septicémie expérimentale de la souris. Comparaison avec la spiramycine base administrée par voie orale.

Experimental septicemia was induced in mice by intraperitoneal injection of 10 to 100 lethal doses of Staphylococcus aureus and Streptococcus pneumoniae. Animals were treated by a mixture of adipic acid and spiramycin (subcutaneous route) or by spiramycin base (oral route), 1 and 6 hours after infection. To determine the effective dose 50% that achieves survival of half the mice after 7 days, each drug was used in 6 dosages (mg/kg) and each dosage was given to 12 mice. In 21 independent experiments, ED50S of spiramycin adipate by the subcutaneous route were found to be 5 to 50 times lower than those of spiramycin base per os. These results are consistent with the high serum peak concentrations of spiramycin adipate observed following subcutaneous administration.