Hyperexpression of the osmB gene in an acidic phospholipid-deficient Escherichia coli mutant

An Escherichia coli pgsA null mutant deficient in acidic phospholipids shows a thermosensitive cell lysis phenotype because of activation of the Rcs phosphorelay signal transduction system. We conducted a DNA microarray analysis with special attention to the genes affected by growth temperature in the mutant deficient in acidic phospholipids. Among the genes identified as highly expressed at high temperature in the pgsA null mutant, the osmB gene was shown to be dependent on the Rcs system for the high expression by dot blot hybridization. Induction of the cloned osmB in the pgsA null mutant caused the thermosensitive defect even in the absence of the Rcs system. Although the deletion of osmB did not suppress the thermosensitivity in the presence of the Rcs system, indicating a multifactorial nature of the deleterious effect of the Rcs activation, we suggest that the osmB hyperexpression is one of the causes of the Rcs-dependent lysis phenotype of the pgsA null mutant.     

RcsA-dependent and -independent growth defects caused by the activated Rcs phosphorelay system in the Escherichia coli pgsA null mutant

In the Escherichia coli pgsA null mutant, which lacks the major acidic phospholipids, the Rcs phosphorelay signal transduction system is activated, causing thermosensitive growth. The mutant grows poorly at 37 degrees C and lyses at 42 degrees C. We showed that the poor growth at 37 degrees C was corrected by disruption of the rcsA gene, which codes for a coregulator protein that interacts with the RcsB response regulator of the phosphorelay system. However, mutant cells still lysed when incubated at 42 degrees C even in the absence of RcsA. We conclude that the activated Rcs phosphorelay in the pgsA null mutant has both RcsA-dependent and -independent growth inhibitory effects. Since the Rcs system has been shown to positively regulate the essential cell division genes ftsA and ftsZ independently of RcsA, we measured cellular levels of the FtsZ protein, but found that the growth defect of the mutant at 42 degrees C did not involve a change in the level of this protein.     

The Cpx two-component signal transduction pathway is activated in Escherichia coli mutant strains lacking phosphatidylethanolamine.

The CpxA-CpxR two-component signal transduction pathway of Escherichia coli was studied in a mutant (pss-93) lacking phosphatidylethanolamine (PE). Several properties of this mutant are comparable to phenotypes of cpxA point mutants, indicating that this two-component pathway is activated in PE-deficient cells. In contrast to point mutants, cpx operon null mutants have a wild-type phenotype. By use of this information, a cpx operon null allele was introduced into a pss-93 mutant. Certain altered properties of PE-deficient mutants, which were consistent with activation of the Cpx pathway, returned to the wild-type phenotype, namely, active accumulation of proline and thiomethyl-beta-D-galactopyranoside was partially restored to wild-type levels, increased resistance to amikacin returned to wild-type sensitivity, and high levels of degP expression returned to repressed wild-type levels. Elevated levels of acetyl phosphate and nlpE gene product can result in activation of the Cpx pathway. However, inactivation of the nlpE gene or mutations eliminating the ability to make acetyl phosphate did not alter the high level of degP expression in pss-93 mutants. We propose that the lack of PE results in an alteration in cell envelope structure or physical properties, leading to direct activation of the Cpx pathway.     

The SixA phospho-histidine phosphatase modulates the ArcB phosphorelay signal transduction in Escherichia coli

The Escherichia coli SixA protein is the first discovered prokaryotic phospho-histidine phosphatase, which was implicated in a His-to-Asp phosphorelay. The sixA gene was originally identified as the one that interferes with, at its multi-copy state, the cross-phosphorelay between the histidine-containing phosphotransmitter (HPt) domain of the ArcB anaerobic sensor and its non-cognate OmpR response regulator. Nevertheless, no evidence has been provided that the SixA phosphatase is indeed involved in a signaling circuitry of the authentic ArcB-to-ArcA phosphorelay in a physiologically meaningful manner. In this study, a SixA-deficient mutant was characterized with special reference to the ArcB signaling, which allows E. coli cells to respond to not only external oxygen, but also certain anaerobic respiratory conditions. Here evidence is provided for the first time that the SixA phosphatase is a crucial regulatory factor that is involved in the ArcB signaling, particularly, under certain anaerobic respiratory growth conditions. We propose a novel mechanism, involving an HPt domain and a phospho-histidine phosphatase, by which a given multi-step His-to-Asp signaling can be modulated.     

Mutations in DnaA protein suppress the growth arrest of acidic phospholipid-deficient Escherichia coli cells

Cell growth arrests when the concentrations of anionic phospholipids drop below a critical level in Escherichia coli, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA-dependent initiation of DNA replication at the chromosomal origin (oriC). Mutations have been introduced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA-acidic phospholipid interactions. Expression of DnaA proteins possessing certain small deletions or substituted amino acids restored growth to cells deficient in acidic phospholipids, whereas expression of wild-type DnaA did not. The mutations include substitutions and deletions in the phospholipid-interacting domain as well as some small deletions in the DNA-binding domain of DnaA. Marker frequency analysis indicated that initiation of replication occurs at or near oriC in acidic phospholipid- deficient cells rescued by the expression of DnaA having a point mutation in the membrane-binding domain, DnaA(L366K). Flow cytometry revealed that expression in wild-type cells of plasmid-borne DnaA(L366K) and DnaA(Delta363-367) reduced the frequency with which replication was initiated and disturbed the synchrony of initiations.     

Cpx two-component signal transduction in Escherichia coli: excessive CpxR-P levels underlie CpxA* phenotypes

In Escherichia coli, the CpxA-CpxR two-component signal transduction system and the sigma(E) and sigma(32) response pathways jointly regulate gene expression in adaptation to adverse conditions. These include envelope protein distress, heat shock, oxidative stress, high pH, and entry into stationary phase. Certain mutant versions of the CpxA sensor protein (CpxA* proteins) exhibit an elevated ratio of kinase to phosphatase activity on CpxR, the cognate response regulator. As a result, CpxA* strains display numerous phenotypes, many of which cannot be easily related to currently known functions of the CpxA-CpxR pathway. It is unclear whether CpxA* phenotypes are caused solely by hyperphosphorylation of CpxR. We here report that all of the tested CpxA* phenotypes depend on elevated levels of CpxR-P and not on cross-signalling of CpxA* to noncognate response regulators.     

Activation of the Rcs signal transduction system is responsible for the thermosensitive growth defect of an Escherichia coli mutant lacking phosphatidylglycerol and cardiolipin

The lethal effect of an Escherichia coli pgsA null mutation, which causes a complete lack of the major acidic phospholipids, phosphatidylglycerol and cardiolipin, is alleviated by a lack of the major outer membrane lipoprotein encoded by the lpp gene, but an lpp pgsA strain shows a thermosensitive growth defect. Using transposon mutagenesis, we found that this thermosensitivity was suppressed by disruption of the rcsC, rcsF, and yojN genes, which code for a sensor kinase, accessory positive factor, and phosphotransmitter, respectively, of the Rcs phosphorelay signal transduction system initially identified as regulating the capsular polysaccharide synthesis (cps) genes. Disruption of the rcsB gene coding for the response regulator of the system also suppressed the thermosensitivity, whereas disruption of cpsE did not. By monitoring the expression of a cpsB'-lac fusion, we showed that the Rcs system is activated in the pgsA mutant and is reverted to a wild-type level by the rcs mutations. These results indicate that envelope stress due to an acidic phospholipid deficiency activates the Rcs phosphorelay system and thereby causes the thermosensitive growth defect independent of the activation of capsule synthesis.     

DjlA negatively regulates the Rcs signal transduction system in Escherichia coli

The Rcs signal transduction system of Escherichia coli regulating capsular polysaccharide synthesis (cps) genes is activated by overexpression of the djlA gene encoding a cytoplasmic membrane-anchored DnaJ-like protein. However, by monitoring the expression of a cpsB'-lac fusion in pgsA- and mdoH-null mutants in which the Rcs system is activated, we found that the Rcs activity was further increased by deletion of djlA and decreased by low-level extrachromosomal expression of djlA. Furthermore, deletion of djlA in a wild-type strain led to small but significant increase of the basal-level activity of the Rcs system. These results demonstrate that DjlA functions as a negative regulator of the Rcs system unless abnormally overproduced.     

Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin

Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability. It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol. However, we now show that a null pgsA mutant is viable, if the major outer membrane lipoprotein is deficient. The pgsA gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids. In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%). Nonetheless, the null mutant grew almost normally in rich media. In low-osmolarity media and minimal media, however, it could not grow. It did not grow at temperatures over 40 degrees C, explaining the previous inability to construct a null pgsA mutant (W. Xia and W. Dowhan, Proc. Natl. Acad. Sci. USA 92:783-787, 1995). Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions. This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in E. coli and explains the suppressing effect of lipoprotein deficiency.     

Transduction of envelope stress in Escherichia coli by the Cpx two-component system.

Disruption of normal protein trafficking in the Escherichia coli cell envelope (inner membrane, periplasm, outer membrane) can activate two parallel, but distinct, signal transduction pathways. This activation stimulates the expression of a number of genes whose products function to fold or degrade the mislocalized proteins. One of these signal transduction pathways is a two-component regulatory system comprised of the histidine kinase CpxA and the response regulator, CpxR. In this study we characterized gain-of-function Cpx* mutants in order to learn more about Cpx signal transduction. Sequencing demonstrated that the cpx* mutations cluster in either the periplasmic, the transmembrane, or the H-box domain of CpxA. Intriguingly, most of the periplasmic cpx* gain-of-function mutations cluster in the central region of this domain, and one encodes a deletion of 32 amino acids. Strains harboring these mutations are rendered insensitive to a normally activating signal. In vivo and in vitro characterization of maltose-binding-protein fusions between the wild-type CpxA and a representative cpx* mutant, CpxA101, showed that the mutant CpxA is altered in phosphotransfer reactions with CpxR. Specifically, while both CpxA and CpxA101 function as autokinases and CpxR kinases, CpxA101 is devoid of a CpxR-P phosphatase activity normally present in the wild-type protein. Taken together, the data support a model for Cpx-mediated signal transduction in which the kinase/phosphatase ratio is elevated by stress. Further, the sequence and phenotypes of periplasmic cpx* mutations suggest that interactions with a periplasmic signaling molecule may normally dictate a decreased kinase/phosphatase ratio under nonstress conditions.     

Inhibition of the signal transduction through the AtoSC system by histidine kinase inhibitors in Escherichia coli

AtoSC two-component system participates in many indispensable processes of Escherichia coli. We report here that the AtoSC signal transduction is inhibited by established histidine kinase inhibitors. Closantel, RWJ-49815 and TNP-ATP belonging to different chemical classes of inhibitors, abrogated the in vitro AtoS kinase autophosphorylation. However, when AtoS was embedded in the membrane fractions, higher inhibitor concentrations were required for total inhibition. When AtoS interacted with AtoC forming complex, the intrinsic histidine kinase was protected by the response regulator, requiring increased inhibitors concentrations for partially AtoS autophosphorylation reduction. The inhibitors exerted an additional function on AtoSC, blocking the phosphotransfer from AtoS to AtoC, without however, affecting AtoC~P dephosphorylation. Their in vivo consequences through the AtoSC inhibition were elucidated on atoDAEB operon expression, which was inhibited only in AtoSC-expressing bacteria where AtoSC was induced by acetoacetate or spermidine. The inhibitor effects were extended on the AtoSC regulatory role on cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis. cPHB was decreased upon the blockers only in acetoacetate-induced AtoSC-expressing cells. Increased ATP amounts during bacterial growth reversed the inhibitory TNP-ATP-mediated effect on cPHB. The alteration of pivotal E. coli processes as an outcome of AtoSC inhibition, establish this system as a target of two-component systems inhibitors.     

Molecular modeling and functional analysis of the AtoS-AtoC two-component signal transduction system of Escherichia coli

The AtoS-AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS-AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS-AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.