pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.     

Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis

Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.     

Catalytic mechanism of S-ribosylhomocysteinase: ionization state of active-site residues

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial autoinducer (AI-2). The proposed catalytic mechanism involves two consecutive ribose carbonyl migration steps via an intramolecular redox reaction and a subsequent beta-elimination step, all catalyzed by a divalent metal ion (e.g., Fe(2+) or Co(2+)) and two general acids/bases in the active site. Absorption and EPR spectroscopic studies were performed with both wild-type and various mutant forms of LuxS under a wide range of pH conditions. The studies revealed a pK(a) of 10.4 for the metal-bound water. The pK(a) value of Cys-83 was determined to be <6 by (13)C-(1)H HSQC NMR experiments with [3-(13)C]cysteine-labeled Zn(2+)-substituted Escherichia coli LuxS. The active form of LuxS contains a metal-bound water and a thiolate ion at Cys-83, consistent with the proposed roles of the metal ion (Lewis acid) and Cys-83 (general acid/base) during catalysis. Finally, an invariant Arg-39 in the active site was demonstrated to be at least partially responsible for stabilizing the thiolate anion of Cys-83.     

Acetate and formate stress: opposite responses in the proteome of Escherichia coli

Acetate and formate are major fermentation products of Escherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-pta strain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of the ackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.     

Expression, export and one-step purification of proteins by fusion to the MalE protein of E. coli

Enzymes can be fused at the C-terminal end of the maltose binding protein (MalE), at the genetic level. Expression of the hybrid proteins, under control of promoter malEp and of the constitutive activator, MalTc1, can be repressed by glucose. The hybrid proteins are localised either in the bacterial cytoplasm or periplasmic space, depending on whether MalE harbors a signal peptide mutation or not; as MalE, they can be purified in one step by chromatography on cross-linked amylose. The Staphylococcus aureus Nuclease and the Klenow portion of E. coli DNA-polymerase I keep their specific activities when fused to MalE.     

Neutralising properties for HIV virus of hybrid protein MalE-CD4 expressed in E. coli and purified in 1 step

Genetic fusions allowing the expression in E. coli of hybrid proteins between a bacterial periplasmic maltose binding protein (MalE) and the CD4 molecule (the receptor of the HIV virus) have been constructed. One of them has kept most of the properties of each constituent: it is exported, can be purified in one step on an affinity column, interacts with anti-MalE and anti-CD4 antibodies, binds HIV gp 120 protein and inactivates HIV virus in an in vitro test.     

Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space

Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose-binding protein of E. coli, or to a signal-sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal-sequence-specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signal-sequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross-linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.     

嗜水气单胞菌群体感应信号分子AI-2的细胞外生物 合成及活性检测

【目的】对嗜水气单胞菌群体感应信号分子AI-2进行细胞外生物合成及活性检测。【方法】对LuxS、MtnN-1、MtnN-2蛋白进行氨基酸序列分析、表达及纯化。以S-腺苷同型半胱氨酸(SAH)为底物,利用纯化的LuxS分别与MtnN-1及MtnN-2蛋白共同作用合成AI-2,并利用哈维氏弧菌报告菌株BB170检测AI-2活性。【结果】嗜水气单胞菌培养液上清中AI-2活性在8 h达到空白对照的16.96倍。氨基酸序列分析表明,嗜水气单胞菌与水生病原菌哈维式弧菌和迟钝爱德华氏LuxS一致性达到76%以上,MtnN-1与MtnN-2氨基酸序列一致性为26.37%,其中MtnN-2与哈维氏弧菌和迟钝爱德华氏菌Pfs一致性达到53%以上。成功表达及纯化了LuxS、MtnN-1和MtnN-2蛋白,细胞外LuxS和MtnN-1共同作用合成的AI-2活性是空白对照的45.04倍,LuxS和MtnN-2共同作用合成的AI-2活性是空白对照的63.62倍。【结论】嗜水气单胞菌能够合成信号分子AI-2。MtnN-1和MtnN-2氨基酸序列尽管存在较大差异,但两者均能与LuxS共同催化AI-2的细胞外生物合成。     

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.     

Crystal structure of a defective folding protein

Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 A resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.     

Synthesis of autoinducer 2 by the lyme disease spirochete, Borrelia burgdorferi

Defining the metabolic capabilities and regulatory mechanisms controlling gene expression is a valuable step in understanding the pathogenic properties of infectious agents such as Borrelia burgdorferi. The present studies demonstrated that B. burgdorferi encodes functional Pfs and LuxS enzymes for the breakdown of toxic products of methylation reactions. Consistent with those observations, B. burgdorferi was shown to synthesize the end product 4,5-dihydroxy-2,3-pentanedione (DPD) during laboratory cultivation. DPD undergoes spontaneous rearrangements to produce a class of pheromones collectively named autoinducer 2 (AI-2). Addition of in vitro-synthesized DPD to cultured B. burgdorferi resulted in differential expression of a distinct subset of proteins, including the outer surface lipoprotein VlsE. Although many bacteria can utilize the other LuxS product, homocysteine, for regeneration of methionine, B. burgdorferi was found to lack such ability. It is hypothesized that B. burgdorferi produces LuxS for the express purpose of synthesizing DPD and utilizes a form of that molecule as an AI-2 pheromone to control gene expression.     

pH-dependent protein profiles of Helicobacter pylori analyzed by two-dimensional gels

Background. Helicobacter pylori survives transient exposure to extreme acid prior to adherence and growth on the gastric epithelium at neutral pH.
Materials and Methods. The effect of pH stress on protein profiles of H. pylori was observed using two-dimensional gel electrophoresis (2-D gels). H. pylori 26695 was grown microaerobically in tryptone-yeast extract broth, 3% fetal bovine serum. Growth in acid alkalinized the medium, whereas growth in base caused acidification. For 2-D gel analysis of protein profiles, cultures were grown in media buffered at pH 5.7 and at pH 7.5.
Results. Under all pH conditions, the most abundant proteins observed were the urease structural subunit UreB and the chaperonin GroEL. Growth in acid significantly increased the abundance of UreB. Thus, urease expression is not completely constitutive, as reported previously, but shows regulation by pH. Another protein observed only at low pH was identified as mammalian apolipoprotein A-I, possibly taken up by H. pylori from bovine serum in the growth medium. This finding, if confirmed, suggests that uptake of high-density lipoprotein from the human host may facilitate acquisition of cholesterol, required for formation of the unique cholesteryl glucosides in the membrane of H. pylori. In growth above pH 7, three stress proteins were induced: GroES (HspA), GroEL (HspB), and the antioxidant AhpC homolog TsaA. In addition, N-terminal sequence analysis identified five additional proteins that had not previously been reported on 2-D gels of H. pylori (FMN, SodB, TrxB, TsaA, and Tsr).
Conclusions. In summary, our 2-D gel study reveals expression of several proteins dependent on growth pH.     

S-Ribosylhomocysteinase (LuxS) is a mononuclear iron protein

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce L-homocysteine and 4,5-dihydroxy-2,3-pentanedione (DHPD). This is a key step in the biosynthetic pathway of the type II autoinducer (AI-2) in both Gram-positive and Gram-negative bacteria. Previous studies demonstrated that LuxS contains a divalent metal cofactor, which has been proposed to be a Zn(2+) ion. To gain insight into the catalytic mechanism of this unusual reaction and the function of the metal cofactor, we developed an efficient expression and purification system to produce LuxS enriched in either Fe(2+), Co(2+), or Zn(2+). Comparison of the catalytic properties and stability of the metal-substituted LuxS with those of the native enzyme revealed that the native metal ion is Fe(2+). The electronic absorption spectrum of the Co(II)-substituted LuxS underwent dramatic catalysis-dependent changes, suggesting the direct involvement of the metal ion in catalysis. Site-directed mutagenesis studies showed that Glu-57 and Cys-84 are essential for catalysis, most likely acting as general acids/bases. Reaction in D(2)O resulted in the incorporation of deuterium at the C-1, C-2, and C-5 positions of the diketone product. These data suggest a catalytic mechanism in which the metal ion catalyzes an intramolecular redox reaction, shifting the carbonyl group from the C-1 position to the C-3 position of the ribose. Subsequent beta-elimination at the C-4 and C-5 positions releases homocysteine as a free thiol.     

A universal system for the transport of redox proteins: early roots and latest developments

The transport of proteins binding redox cofactors across a biological membrane is complicated by the fact that insertion of the redox cofactor is often a cytoplasmic process. These cytoplasmically assembled redox proteins must thus be transported in partially or completely folded form. The need for a special transport system for redox proteins was first recognized for periplasmic hydrogenases in gram-negative bacteria. These enzymes, which catalyze the reaction H2 <--> 2H+ + 2e, are composed of a large and a small subunit. Only the small subunit has an unusually long signal sequence of 30-50 amino acid residues, characterized by a conserved motif (S/T)-R-R-x-F-L-K at the N-terminus. This sequence directs export of the large and small subunit complex to the periplasm. Sequencing of microbial genes and genomes has shown that signal sequences with this conserved motif, now referred to as twin-arginine leaders, occur ubiquitously and export different classes of redox proteins, containing iron sulfur clusters, molybdopterin cofactors, polynuclear copper sites or flavin adenine dinucleotide. Mutations in an Escherichia coli operon referred to as mtt (membrane targeting and translocation) or tat (twin arginine translocation) are pleiotropic, i.e. these prevent the expression of a variety of periplasmic oxido-reductases in functional form. The Mtt or Tat pathway is distinct from the well-known Sec pathway and occurs ubiquitously in prokaryotes. The fact that its component proteins share sequence homology with proteins of the delta pH pathway for protein transport associated with chloroplast thylakoid assembly, illustrates the universal nature of this novel protein translocation system.     

水稻内生成团泛菌YS19共质体形成差异表达蛋白MalE及其兼职功能

成团泛菌YS19是从水稻“越富”品种中分离的一种优势内生细菌,与宿主水稻互作时具有多种促生作用,其形成的共质体(symplasmata)结构与菌体抗逆及与宿主互作有重要意义．研究发现了一种在YS19共质体形成阶段高表达的差异蛋白,对其用肽指纹图谱进行鉴定,发现其属于周质空间麦芽糖结合蛋白家族．克隆了该蛋白质的基因,重组表达并分离纯化了该蛋白质,发现它是一种兼职功能蛋白,其不仅参与麦芽糖的ABC运输系统,而且在强酸环境下不易发生变性沉淀,并可通过疏水面的显著暴露结合底物蛋白来发挥分子伴侣活性,这些兼职功能构成了菌体抗逆生存适应性的重要分子基础．     

Raising antibodies against OprD, an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE.

OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).