Hydrolysis of peptidoglycan is modulated by amidation of meso‐diaminopimelic acid and Mg2+ in Bacillus subtilis

The ability of excess Mg²⁺ to compensate the absence of cell wall related genes in Bacillus subtilis has been known for a long time, but the mechanism has remained obscure. Here, we show that the rigidity of wild‐type cells remains unaffected with excess Mg²⁺, but the proportion of amidated meso‐diaminopimelic (mDAP) acid in their peptidoglycan (PG) is significantly reduced. We identify the amidotransferase AsnB as responsible for mDAP amidation and show that the gene encoding it is essential without added Mg²⁺. Growth without excess Mg²⁺ causes ΔasnB mutant cells to deform and ultimately lyse. In cell regions with deformations, PG insertion is orderly and indistinguishable from the wild‐type. However, PG degradation is unevenly distributed along the sidewalls. Furthermore, ΔasnB mutant cells exhibit increased sensitivity to antibiotics targeting the cell wall. These results suggest that absence of amidated mDAP causes a lethal deregulation of PG hydrolysis that can be inhibited by increased levels of Mg²⁺. Consistently, we find that Mg²⁺ inhibits autolysis of wild‐type cells. We suggest that Mg²⁺ helps to maintain the balance between PG synthesis and hydrolysis in cell wall mutants where this balance is perturbed in favor of increased degradation.     

Rhythmic Fluctuations in the Concentration of Intracellular Mg2+ in Association with Spontaneous Rhythmic Contraction in Cultured Cardiac Myocytes

Magnesium ions (Mg²⁺) play a fundamental role in cellular function, but the cellular dynamic changes of intracellular Mg²⁺ remain poorly delineated. The present study aims to clarify whether the concentration of intracellular Mg²⁺ possibly changes cyclically in association with rhythmic contraction and intracellular Ca²⁺ oscillation in cultured cardiac myocytes from neonatal rats. To do this, we performed a noise analysis of fluctuations in the concentration of intracellular Mg²⁺ in cardiac myocytes. The concentration was estimated by loading cells with either Mg‐fluo4/AM or KMG‐20/AM. Results revealed that the intensity of Mg‐fluo‐4 or KMG‐20 fluorescence fluctuated cyclically in association with the rhythmic contraction of cardiac myocytes. In addition, the simultaneous measurement of Fura2 and Mg‐fluo‐4 fluorescence revealed phase differences between the dynamics of the two signals, suggesting that the cyclic changes in the Mg‐fluo‐4 or KMG‐20 fluorescent intensity actually reflected the changes in intracellular Mg²⁺. The complete termination of spontaneous rhythmic contractions did not abolish Mg²⁺ oscillations, suggesting that the rhythmic fluctuations in intracellular Mg²⁺ did not result from mechanical movements. We suggest that the concentration of intracellular Mg²⁺ changes cyclically in association with spontaneous, cyclic changes in the concentration of intracellular Ca²⁺ of cardiac myocytes. A noise analysis of the fluctuation of subtle changes in fluorescence intensity could contribute to the elucidation of novel functional roles of Mg²⁺ in cells.     

Imipramine inhibition of TRPM‐like plasmalemmal Mg2+ transport in vascular smooth muscle cells

Depression is associated with vascular disease, such as myocardial infarction and stroke. Pharmacological treatments may contribute to this association. On the other hand, Mg²⁺ deficiency is also known to be a risk factor for the same category of diseases. In the present study, we examined the effect of imipramine on Mg²⁺ homeostasis in vascular smooth muscle, especially via melastatin‐type transient receptor potential (TRPM)‐like Mg²⁺‐permeable channels. The intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured using ³¹P‐nuclear magnetic resonance (NMR) in porcine carotid arteries that express both TRPM6 and TRPM7, the latter being predominant. pH_i and intracellular phosphorus compounds were simultaneously monitored. To rule out Na⁺‐dependent Mg²⁺ transport, and to facilitate the activity of Mg²⁺‐permeable channels, experiments were carried out in the absence of Na⁺ and Ca²⁺. Changing the extracellular Mg²⁺ concentration to 0 and 6 mM significantly decreased and increased [Mg²⁺]_i, respectively, in a time‐dependent manner. Imipramine statistically significantly attenuated both of the bi‐directional [Mg²⁺]_i changes under the Na⁺‐ and Ca²⁺‐free conditions. This inhibitory effect was comparable in influx, and much more potent in efflux to that of 2‐aminoethoxydiphenyl borate, a well‐known blocker of TRPM7, a channel that plays a major role in cellular Mg²⁺ homeostasis. Neither [ATP]_i nor pH_i correlated with changes in [Mg²⁺]_i. The results indicate that imipramine suppresses Mg²⁺‐permeable channels presumably through a direct effect on the channel domain. This inhibitory effect appears to contribute, at least partially, to the link between antidepressants and the risk of vascular diseases.     

Site‐Selective In Situ Electrochemical Doping for Mn‐Rich Layered Oxide Cathode Materials in Lithium‐Ion Batteries

Various doped materials have been investigated to improve the structural stability of layered transition metal oxides for lithium‐ion batteries. Most doped materials are obtained through solid state methods, in which the doping of cations is not strictly site selective. This paper demonstrates, for the first time, an in situ electrochemical site‐selective doping process that selectively substitutes Li⁺ at Li sites in Mn‐rich layered oxides with Mg²⁺. Mg²⁺ cations are electrochemically intercalated into Li sites in delithiated Mn‐rich layered oxides, resulting in the formation of [Li_1?xMg_y][Mn_1?zM_z]O₂ (M = Co and Ni). This Mg²⁺ intercalation is irreversible, leading to the favorable doping of Mg²⁺ at the Li sites. More interestingly, the amount of intercalated Mg²⁺ dopants increases with the increasing amount of Mn in Li_1?x[Mn_1?zM_z]O₂, which is attributed to the fact that the Mn‐to‐O electron transfer enhances the attractive interaction between Mg²⁺ dopants and electronegative O^δ? atoms. Moreover, Mg²⁺ at the Li sites in layered oxides suppresses cation mixing during cycling, resulting in markedly improved capacity retention over 200 cycles. The first‐principle calculations further clarify the role of Mg²⁺ in reduced cation mixing during cycling. The new concept of in situ electrochemical doping provides a new avenue for the development of various selectively doped materials.     

Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation

Abstract. Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium. Objectives: Here, we have investigated the roles of melastatin‐like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca²⁺) and magnesium (Mg²⁺) conducting channels, during proliferation of human osteoblasts. Results: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast‐like MG‐63, SaOS and U2‐OS cells, and reduction of extracellular Mg²⁺ or Ca²⁺ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg²⁺ and Ca²⁺ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre‐incubation of cells in reduced extracellular Mg²⁺ conditions led to activation of Ca²⁺ and Mg²⁺ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation. Conclusions: Our results indicate that extracellular Mg²⁺ and Ca²⁺ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg²⁺ and Ca²⁺ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.     

Erythrocytes anion transport and oxidative change in β‐thalassaemias

β‐Thalassaemia is characterized by a decrease in globin β‐chain synthesis and an excess in free α‐globin chains. This induces alterations in membrane lipids and proteins resulting from a reduction in spectrin/band 3 ratio, partial oxidation of band 4.1 and clustering of band 3. The membrane injury provokes hyperhaemolysis and bone marrow hyperplasia. The pathophysiology of thalassaemia is associated with iron overload that generates oxygen free radicals and oxidative tissue injury with ocular vessel alterations. The aim of this research is to investigate the influence of oxidative stress on band 3 efficiency, which is an integral membrane protein of RBCs (red blood cells). Band 3 protein, of which there are more than 1 million copies per cell, is the most abundant membrane protein in human RBCs. It mediates the anion exchange and acid–base equilibrium through the RBC membrane. Some experiments were performed on thalassaemic cells and β‐thalassaemia‐like cells and tested for sulfate uptake. To test the antioxidant effect of Mg²⁺, other experiments were performed using normal and pathological cells in the presence of Mg²⁺. The oxidant status in thalassaemic cells was verified by increased K⁺ efflux, by lower GSH levels and by increased G6PDH (glucose‐6‐phosphate dehydrogenase) activity. The rate constant of SO₄ ^2? uptake decreases in thalassaemic cells as well as in β‐thalassaemia‐like cells when compared with normal cells. It increases when both cells are incubated with Mg²⁺. Our data show that oxidative stress plays a relevant role in band 3 function of thalassaemic cells and that antioxidant treatment with Mg²⁺ could reduce oxidative damage to the RBC membrane and improve the anion transport efficiency regulated by band 3 protein.     

Cadmium accumulation and its effects on intracellular ion pools in a brewing strain of Saccharomyces cerevisiae

The presence of glucose resulted in a two- to three-fold increase in levels of Cd²⁺accumulated by Saccharomyces cerevisiae after 5 h compared with those observed in the absence of glucose. However, time-dependent Cd²⁺ uptake continued in the absence of glucose over 5 h, resulting in an appreciable increase in cellular Cd²⁺levels. Substantial K⁺ efflux but little Mg²⁺ and negligible Ca²⁺release was observed. Cell fractionation revealed that the bulk of intracellular Cd²⁺ was located in the vacuolar (25%) and bound (60%) fractions. Accumulation of Cd²⁺ ions impacted most noticeably on K⁺ rather than Mg²⁺ levels in intracellular compartments. Cytoplasmic and particularly vacuolar K⁺ levels decreased as Cd²⁺ sequestration continued resulting in increased extracellular levels. In contrast, corresponding intracellular Mg²⁺ pools were only modestly affected with a slight increase and decrease observed in the cytoplasmic and vacuolar fractions respectively. However, levels of bound Mg²⁺ decreased in response to continued Cd²⁺ accumulation. Received 07 March 1999/ Accepted in revised form 26 June 1999     

Time course of antioxidant responses of Capsicum annuum subjected to a progressive magnesium deficiency

The effect of magnesium (Mg²⁺)‐deficiency on the antioxidant responses of Capsicum annuum was investigated over a 60‐day period under controlled conditions. This Mg²⁺‐deficiency aimed to mimic the physiological conditions that plants may experience in the field. At each harvest time, five different leaf‐levels (L2 to L6) were distinguished. L2 and L6 correspond to the second and sixth youngest leaves, respectively. The following parameters were determined: Mg²⁺, chlorophyll and protein contents, total and redox pools of ascorbate and glutathione, and the activities of superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Under Mg²⁺‐deficiency, leaf Mg²⁺ contents decreased over time in all leaf‐levels except in the second youngest leaves (L2), where they remained constant at about 0.25% (dry weight basis). Mg²⁺‐deficiency led to an increase in the antioxidant enzyme activities concomitant with an increase in the ascorbate and glutathione pools, whereas total chlorophyll and soluble protein contents decreased. The L2 leaves showed an increase in glutathione reductase activity and in the ascorbate redox state whereas no difference was observed for the other parameters. Superoxide dismutase activities increased in L5 leaves from day 15 and, afterwards, in L3 to L5 leaves, irrespective of Mg²⁺ content. At day 30, glutathione reductase activities increased in L2 to L4 leaves and dehydroascorbate reductase activities in L4 leaves. At day 45, we observed an increase in the ascorbate peroxidase activities in L3 to L5 leaves. At the same time, ascorbate and glutathione pools increased in intermediate leaves, whereas chlorophyll content decreased in L3 and L4 leaves, and protein content decreased in L4 leaves. Results suggest that pepper leaves enhance their defence capacities against oxidative stress by increasing ascorbate more than glutathione synthesis. However, cells showed higher regeneration rates for the glutathione redox state than for the ascorbate redox state.     

Mg2+‐dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis

The MgtE family of Mg²⁺ transporters is ubiquitously distributed in all phylogenetic domains. Recent crystal structures of the full‐length MgtE and of its cytosolic domain in the presence and absence of Mg²⁺ suggested a Mg²⁺‐homeostasis mechanism, in which the MgtE cytosolic domain acts as a ‘Mg²⁺ sensor’ to regulate the gating of the ion‐conducting pore in response to the intracellular Mg²⁺ concentration. However, complementary functional analyses to confirm the proposed model have been lacking. Moreover, the limited resolution of the full‐length structure precluded an unambiguous characterization of these regulatory divalent‐cation‐binding sites. Here, we showed that MgtE is a highly Mg²⁺‐selective channel gated by Mg²⁺ and elucidated the Mg²⁺‐dependent gating mechanism of MgtE, using X‐ray crystallographic, genetic, biochemical, and electrophysiological analyses. These structural and functional results have clarified the control of Mg²⁺ homeostasis through cooperative Mg²⁺ binding to the MgtE cytosolic domain.     

Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (¹O₂) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to ¹O₂. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with ¹O₂ or superoxide (O^–₂). One sRNA was specifically induced by ¹O₂ and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by ¹O₂ and O^–₂ were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH_I and RpoH_II. The most abundant sRNA was processed in the presence of ¹O₂ but not by O^–₂. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of ¹O₂‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by ¹O₂ exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.     

Elevating intracellular free Mg2+ preserves sensitivity of Na+−K+ pump to ATP at reduced temperatures in guinea pig red blood cells

Red cells of hibernating species have a higher relative rate of Na⁺–K⁺ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na⁺–K⁺ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37°C by measuring ouabain-sensitive K⁺ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg²⁺ to 2 mmol·l^-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20°C, rather than to decrease, as occurs in cells not loaded with Mg²⁺. In ground squirrel cells raising intracellular free Mg²⁺ had little effect on apparent affinity of the pump for ATP at 20°C. ATP affinity rose slightly with cooling both in Mg²⁺-enriched and in control ground squirrel cells. Increased intracellular free Mg²⁺ in guinea pig cells stimulated Na⁺–K⁺ pump activity so that at 20°C the pump rate was the same in the Mg²⁺-enriched guinea pig and control ground squirrel cells. Pump activity in Mg²⁺-enriched guinea pig cells at 5°C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na⁺–K⁺ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg²⁺. Conversely, in ground squirrel red cells natural rise of free Mg²⁺ may in part account for the preservation of the ATP affinity of their Na⁺–K⁺ pump with cooling.Abbreviations K _m Michaelis-Menten constant for apparent affinity - MOPS 3-(N-morpholino)-propanesulphonic acid - [Mg²⁺]_i intracellular concentration of free Mg²⁺ - OD optical density - RBC red blood cell(s) - T _b body temperature     

Control of membrane potential and excitability ofChara cells with ATP and Mg2+

Summmary Electric characteristics of internodalChara australis cells, from which the tonoplast had been removed by vacuolar perfusion with media containing EGTA, were studied in relation to intracellular concentrations of ATP and Mg²⁺ using the ordinary microelectrode method and the open-vacuole method developed by Tazawa, Kikuyama and Nakagawa (1975.Plant Cell Physiol. 16:611). The concentration of ATP was decreased by introducing hexokinase and glucose into the cell and that of Mg²⁺ by introducing EDTA or CyDTA. The membrane potential decrease and the membrane resistance increase were both significant when the ATP or Mg²⁺ concentration was decreased. An ATP-dependent membrane potential was also found in other species of Characeae,Nitella axillaris andN. pulchella. Excitability of the membrane was also completely lost by reducing the ATP or Mg²⁺ concentration. Both membrane potential and excitability were recovered by introducing ATP or Mg²⁺ into ATP- or Mg²⁺-depleted cells.The time course of membrane potential recovery was followed by the open-vacuole method. Recovery began as soon as intracellular perfusion with medium containing ATP and Mg²⁺ was started. Reversible transition of the membrane potential between polarized and pepolarized levels by controlling the intracellular concentration of ATP or Mg²⁺ could be repeated many times by the open-vacuole method, when the excitability was suppressed by addition of Pb²⁺ to the external medium.The ineffectiveness of an ATP analog, AMP-PNP, and the synergism of ATP and Mg²⁺ in maintaining the membrane potential and excitability strongly suggest that ATP act via its hydrolysis by Mg²⁺-activated ATPase. The passive nature of the membrane, as judged from responses of the membrane potential to changes of the external K⁺ concentration, was not altered by lowering the ATP concentration in the cell. The mechanism of membrane potential generation dependent on ATP is discussed on the basic of an electrogenic ion pump. Involvement of the membrane potential generated by the ion pump in the action potential is also discussed.     

Magnesium-Dependent stimulation of protein synthesis by the insulin mimic,pervanadate

The insulin mimic, peroxide of vanadate (pervanadate), stimulated ³⁵S-methionine incorporation into Xenopus oocyte protein in a Mg²⁺-dependent manner. Reducing the extracellular Mg²⁺ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg²⁺ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg²⁺ to 0.01 mM inhibited about 50% of the insulinstimulated component of methionine incorporation. Mg²⁺ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn²⁺ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg²⁺. Reducing extracellular Ca²⁺ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 m?I droplet of buffer containing the Mg²⁺ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg²⁺ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg²⁺. In contrast, apparent extracellular Mg²⁺ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg²⁺, but not Ca²⁺, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg²⁺, and this change in membrane transport may be an early event in signalling the increase in translation. © 1995 Wiley-Liss, Inc.     

V‐ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2C

The activity of vacuolar H⁺‐ATPase (V‐ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5‐HT). 5‐HT induces, via protein kinase A, the phosphorylation of V‐ATPase subunit C and the assembly of V‐ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V‐ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK‐506) do not prevent V‐ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg²⁺ level caused by loading secretory cells with EDTA‐AM leads to the activation of proton pumping in the absence of 5‐HT, prolongs the 5‐HT‐induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V‐ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg²⁺, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.     

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N‐terminal Fic domain and a C‐terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS‐mediated translocation into host cells. A proteolysis resistant fragment (residues 10–302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α‐[³²P]‐ATP. Its crystal structure, determined to 2.9‐Å resolution by the SeMet‐SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β‐rich domain at the C‐terminus. On crystal soaking with ATP/Mg²⁺, additional electron density indicated the presence of a PP_i/Mg²⁺ moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg²⁺ and target tyrosine. The model is consistent with an in‐line nucleophilic attack of the deprotonated side‐chain hydroxyl group onto the α‐phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence‐independent mechanism of target positioning through antiparallel β‐strand interactions between enzyme and target is suggested.     

MicA sRNA links the PhoP regulon to cell envelope stress

Numerous small RNAs regulators of gene expression exist in bacteria. A large class of them binds to the RNA chaperone Hfq and act by base pairing interactions with their target mRNA, thereby affecting their translation and/or stability. They often have multiple direct targets, some of which may be regulators themselves, and production of a single sRNA can therefore affect the expression of dozens of genes. We show in this study that the synthesis of the Escherichia coli pleiotropic PhoPQ two‐component system is repressed by MicA, a σ^E‐dependent sRNA regulator of porin biogenesis. MicA directly pairs with phoPQ mRNA in the translation initiation region of phoP and presumably inhibits translation by competing with ribosome binding. Consequently, MicA downregulates several members of the PhoPQ regulon. By linking PhoPQ to σ^E, our findings suggest that major cellular processes such as Mg²⁺ transport, virulence, LPS modification or resistance to antimicrobial peptides are modulated in response to envelope stress. In addition, we found that Hfq strongly affects the expression of phoP independently of MicA, raising the possibility that even more sRNAs, which remain to be identified, could regulate PhoPQ synthesis.