Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli

Escherichia coli is the most widely used host for producing membrane proteins. Thus far, to study the consequences of membrane protein overexpression in E. coli, we have focussed on prokaryotic membrane proteins as overexpression targets. Their overexpression results in the saturation of the Sec translocon, which is a protein-conducting channel in the cytoplasmic membrane that mediates both protein translocation and insertion. Saturation of the Sec translocon leads to (i) protein misfolding/aggregation in the cytoplasm, (ii) impaired respiration, and (iii) activation of the Arc response, which leads to inefficient ATP production and the formation of acetate. The overexpression yields of eukaryotic membrane proteins in E. coli are usually much lower than those of prokaryotic ones. This may be due to differences between the consequences of the overexpression of prokaryotic and eukaryotic membrane proteins in E. coli. Therefore, we have now also studied in detail how the overexpression of a eukaryotic membrane protein, the human KDEL receptor, affects E. coli. Surprisingly, the consequences of the overexpression of a prokaryotic and a eukaryotic membrane protein are very similar. Strain engineering and likely also protein engineering can be used to remedy the saturation of the Sec translocon upon overexpression of both prokaryotic and eukaryotic membrane proteins in E. coli.     

Effect of benzoic acid hydroxyl- and methoxy-ring substituents on cucumber (Cucumis sativus L.) root membrane potential

Abstract

The allelopathic effect of some benzoic acid (BA) OH- and OCH₃-ring substituents was studied on cucumber root transmembrane potential difference (Vm). Most of the methoxy-BAs induced a rapid Vm depolarization, followed by a Vm hyperpolarization, with the only exception for p-anisic acid (pA). On the other hand, salicylic acid (SA) and 3,4-dimethoxybenzoic acid (DHB) strongly depolarized Vm. A positive correlation was found between Vm hyperpolarization and lipophilicity of methoxylated BAs, whereas a positive correlation was found between lipophilicity and Vm depolarization of hydroxylated BAs. The influence of BAs on K⁺ was studied by means of specific blocking with Cs⁺ indicating a possible direct interaction of SA, gallic acid (GA), vanillic acid (VA) and 3,4-dimethoxybenzoic acid (DMB). Interference of BAs with the Vm hyperpolarizing effect of root perfusion with the fungal toxin fusicoccin were also observed.     

Characterization of a potassium carrier in rabbit reticulocyte cell membrane

Summary In this study we present evidence that high ouabain-resistant Rb influx, carried out by the rabbit reticulocyte membrane, is composed of carrier-mediated Rb influx and passive diffusion across the cell membrane. To meet this end, an assay was developed by which the two ouabain-resistant Rb influxes could be measured separately.Whereas theK _m for Rb of the carrier (12.5mm) did not change by increasing the pH, theVm was markedly reduced. At the optimal pH (6.0–6.5) theVm was 6–8 mmol h^–1 liter^–1 and fell to zero at pH 8.0. This may indicate a possible role of H⁺ ions in this transport mechanism.The carrier is inhibited by furosemide and ethacrynic acid, similarly to pump II in the erythrocyte and kidney. In addition, its activity is dependent upon the ionic content of the medium. The K(Rb) carrier appeared not to be involved in an active transport since depletion of ATP had no effect on the carrier activity. The carrier activity was also measured in rabbit erythrocytes and was found to be 10 times lower than that of rabbit reticulocytes. TheK _m for Rb, optimal pH, and high sensitivity to furosemide and ethacrynic acid of the erythrocyte and the reticulocyte carrier are similar.Our study suggests that maturation of reticulocytes to erythrocytes is accompanied by a loss or inactivation of most of a K (or Rb) carrier very active in the reticulocyte cell.     

The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus Aspergillus flavus

As a pathogenic fungus, Aspergillus flavus can produce carcinogenic aflatoxins (AFs), which poses a great threat to crops and animals. Msb2, the signalling mucin protein, is a part of mitogen-activated protein kinase (MAPK) pathway which contributes to a range of physiological processes. In this study, the roles of membrane mucin Msb2 were explored in A. flavus by the application of gene disruption. The deletion of msb2 gene (Δmsb2) caused defects in vegetative growth, sporulation and sclerotia formation when compared to WT and complement strain (Δmsb2^C) in A. flavus. Using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis, it was found that deletion of msb2 down-regulated aflatoxin B₁ (AFB₁) synthesis and decreased the infection capacity of A. flavus. Consistently, Msb2 responds to cell wall stress and osmotic stress by positively regulating the phosphorylation of MAP kinase. Notably, Δmsb2 mutant exhibited cell wall defect, and it was more sensitive to inhibitor caspofungin when compared to WT and Δmsb2^C. Taking together, these results revealed that Msb2 plays key roles in morphological development process, stresses adaptation, secondary metabolism and pathogenicity in fungus A. flavus.     

Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris

The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate‐screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N‐methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing “jackpot” clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in‐culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.     

Metabolic shutdown in Escherichia coli cells lacking the outer membrane channel TolC

The outer membrane channel TolC is a key component of multidrug efflux and type I secretion transporters in Escherichia coli. Mutational inactivation of TolC renders cells highly susceptible to antibiotics and leads to defects in secretion of protein toxins. Despite impairment of various transport functions, no growth defects were reported in cells lacking TolC. Unexpectedly, we found that the loss of TolC notably impairs cell division and growth in minimal glucose medium. The TolC‐dependent phenotype was further exacerbated by the loss of ygiB and ygiC genes expressed in the same operon as tolC and their homologues yjfM and yjfC located elsewhere on the chromosome. Our results show that this growth deficiency is caused by depletion of the critical metabolite NAD⁺ and high NADH/NAD⁺ ratios. The increased amounts of PspA and decreased rates of NADH oxidation in ΔtolC membranes indicated stress on the membrane and dissipation of a proton motive force. We conclude that inactivation of TolC triggers metabolic shutdown in E. coli cells grown in minimal glucose medium. The ΔtolC phenotype is partially rescued by YgiBC and YjfMC, which have parallel functions independent from TolC.     

Effect of vinblastine sulfate, colchicine and lumicolchicine on membrane organization of normal and transformed cells

Differences in the distribution of plasma membrane intramembranous particles (PMP) have been demonstrated in normal and transformed fibroblasts using freeze fracture and electron microscopy. Transformed 3T3 cells contain randomly distributed PMP and contact-inhibited 3T3 cells have aggregated PMP when frozen in medium, glycerol, sucrose, or following stabilization in 1 % formaldehyde. To define some of the mechanisms controlling the organization of PMP in this system we have examined the effects of microtubule disruptive drugs including vinblastine sulfate and colchicine on SV3T3 cells. These drugs were observed to induce a dose- and time-dependent aggregation of PMP at concentrations between 10⁻⁹ and 10⁻⁵ M. These results suggest that modulation of PMP distribution in these cells may be influenced by an interaction of microtubules with plasma membrane components. However, the observation that lumicolchicine, a derivative of colchicine which does not disrupt microtubules, also promotes PMP aggregation, suggests that these drugs may also have a primary effect on the plasma membrane in addition to the disruption of microtubules. This is supported by the observation that reduced temperature (4 °C) which is known to disrupt microtubules fails to induce PMP aggregation in SV3T3 cells, suggesting the hypothesis that changes in the interaction of plasma membrane or plasma membrane associated constituents may control the distribution of PMP in this cell system.     

Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

Background  

The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRβ subunit is an integral membrane protein, which tethers the SRP-interacting SRα subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRβ subunit and consists of only the SRα homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting.     

Enhanced exudation of malate in the rhizosphere due to AtALMT1 overexpression in blackgram (Vigna mungo L.) confers increased aluminium tolerance

• Worldwide, 50% of soil is acidic, which induces aluminium (Al) toxicity in plants, as the phyto‐availability of Al³⁺ increases in acidic soil. Plants responds to Al³⁺ toxicity by exuding organic acids into the rhizosphere. The organic acid responsible for Al³⁺ stress response varies from species to species, which in the case of blackgram (Vigna mungo L.) is citrate.
• In blackgram, an Arabidopsis malate transporter, AtALMT1, was overexpressed with the motive of inducing enhanced exudation of malate. Transgenics were generated using cotyledon node explants through Agrobacterium tumefaciens‐mediated transformation. The putative transgenics were initially screened by AtALMT1‐specific genomic DNA PCR, followed by quantitative PCR. Two independent transgenic events were identified and functionally characterized in the T₃ generation.
• The transgenic lines, Line 1 and 2, showed better root growth, relative water content and chlorophyll content under Al³⁺ stress. Both lines also accounted for less oxidative damage, due to reduced accumulation of ROS molecules. Photosynthetic efficiency, as measured in terms of F_v/F_m, NPQ and Y(II), increased when compared to the wild type (WT). Relative expression of genes (VmSTOP1, VmALS3, VmMATE) responsible for Al³⁺ stress response in blackgram showed that overexpression of a malate transporter did not have any effect on their expression. Malate exudation increased whereas citrate exudation did not show any divergence from the WT. A pot stress assay found that the transgenics showed better adaptation to acidic soil.
• This report demonstrates that the overexpression of a malate transporter in a non‐malate exuding species improves adaptation to Al³⁺ toxicity in acidic soil without effecting its stress response mechanism.

     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

Vm-milR21调控苹果黑腐皮壳侵染致病的功能和机理

苹果黑腐皮壳(Valsamali)引起的腐烂病是苹果最具毁灭性的枝干病害。微小核糖核酸(microRNA-like RNAs, milRNAs)在真菌生长发育、侵染致病和胁迫响应等过程中发挥重要作用。前期研究发现Vm-milR21在病菌侵染阶段特异性下调表达,推测其可能参与V. mali的侵染致病过程。【目的】本文对Vm-milR21的功能进行研究。【方法】制备Vm-milR21前体过表达和沉默转化株,评价不同转化株与野生型菌株表型差异。进而,通过实时荧光定量聚合酶链反应(quantitative real time polymerase chain reaction, qRT-PCR)和共转化技术验证Vm-milR21与其潜在靶标Vm-03494之间的靶向调控关系。在此基础上,构建Vm-03494的基因敲除突变体,并鉴定突变体表型。【结果】与野生型菌株相比,过表达转化株的菌丝生长速率以及对叶片和枝条的致病力均大幅降低,而沉默转化株无明显变化。Vm-milR21能够序列特异性地抑制Vm-03494表达。相较于野生型菌株,Vm-03494敲除突变体菌丝生长速率和致病力均显著降低。【结论】...     

Short-term effects of plant hormones on membrane potential and membrane permeability of dwarf maize coleoptile cells (Zea mays L. d 1) in comparison with growth responses

The membrane potential difference of dwarf maize coleoptile cells is increased by both 10^-5moll^-1 gibberellic acid (GA₃) and indoleacetic acid (IAA) a few minutes after application. A final level is reached after 10–20 min. The membrane permeability ratio P _Na:P _K is altered by both hormones during the first 15 min after application, indicating a rapid effect on the membrane. Elongation growth of coleoptile segments, however, is only stimulated by IAA. The auxin-induced growth as well as the auxin effect on membrane permeability depends on the calcium ion concentration of the medium. It is concluded that IAA acts via a proton extrusion pump that is electrically balanced by a potassium ion uptake, driven by the electromotive force of the pump. The mode of action of GA₃ on elongation growth is assumed to involve a process that depends on the physiologic state of the tissue and/or metabolic energy.Abbreviations IAA indoleacetic acid - GA₃ gibberellic acid - FC fusicoccin - PD electric potential difference between the vacuole and the external medium     

A study of dielectric membrane breakdown in theFucus egg

Summary Unfertilized eggs and zygotes of the marine brown alga,Fucus serratus, have been subjected to single external electric field pulses of 1 to 1760 sec duration ( _p ) and 50 to 400 V field strength (U _p cm^–1). During exposure, the difference in electric potential across the plasmalemma (V _m ) was recorded intracellularly from single eggs, and the efflux of⁸⁶Rb⁺(K⁺) from the cytoplasm was measured on egg populations. A given single pulse instantaneously depolarizes the plasmalemma by a few (i.e., 6) millivolts and releases a certain fraction (i.e., 5%) of the cytoplasmic⁸⁶Rb⁺(K⁺). The dependence of these responses uponU _p and _p is fully consistent with the assumption that the membrane undergoes a localized reversible dielectric breakdown and reseals within <3 sec. The data are treated in terms of the electro-mechanical model for a compressible membrane by H.G.L. Coster and U. Zimmermann (1975,J. Membrane Biol. 22:73) and verify this model on a nonvacuolated plant cell. A thresholdV _m for membrane breakdown (V _c ) of 0.58 and 0.51 V is estimated for the turgorless undertilized eggs and the turgescent (4.8 bar) zygotes, respectively. Using these values forV _c , and a reasonable value of the membrane's elastic modulus (i.e.,Y _m 10⁶ Nm^–2), possible sites of membrane breakdown are dicussed in terms of membrane thickness and relative permittivity.     

Kinetics of Ca2+- and ATP-dependent, voltage-controlled anion conductance in the plasma membrane of mesophyll cells of Pisum sativum

Whole-cell patch-clamp techniques were used to measure anion currents through the plasma membrane of protoplasts of mesophyll cells of expanding pea (Pisum sativum L.) leaves. Voltage-induced changes of the currents could be modelled with single exponential activation and deactivation kinetics. The anion currents were activated at negative membrane potentials. The time constant of activation, τ_act, increased from 145 ms at −140 mV to 380 ms at −20 mV. A Boltzmann fit to the activation curve, n_∞ (ΔG_Vm/ΔG_max), yielded a half-activation voltage of +27 mV. Opening and closing rate constants, α and β respectively, were calculated from the values of τ and n_∞. The currents depended on the presence of cytoplasmic Ca²⁺ concentrations higher than 10⁻⁶ M. Including 3 mM MgATP in the intracellular solution resulted in a voltage-dependent inactivation of the anion current. The conductance-voltage relation resulting from the voltage-dependent activation and inactivation had a maximum at about −25 mV. The relations of the current in pea are discussed with respect to the anion currents in guard cells and suspension-cultured tobacco cells, and its possible role in growing leaf cells. Received: 1 March 1996 / Accepted: 16 September 1996     

Characterization of<Emphasis Type="Italic"> Schizosaccharomyces pombe</Emphasis> mutants defective in vacuolar acidification and protein sorting

The vacuolar H⁺-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. To investigate the functional roles of the V-ATPase in Schizosaccharomyces pombe, the gene vma1 encoding subunit A or vma3 encoding subunit c was disrupted. Both deletion mutants lost the capacity for vacuolar acidification in vivo, and showed sensitivity to neutral pH or high concentrations of divalent cations including Ca²⁺. The delivery of FM4-64 to the vacuolar membrane and accumulation of Lucifer Yellow CH were strongly inhibited in the vma1 and vma3 mutants. Moreover, deletion of the S. pombe vma1 ⁺ or vma3 ⁺ gene resulted in pleiotropic phenotypes consistent with lack of vacuolar acidification, including the missorting of vacuolar carboxypeptidase Y, abnormal vacuole morphology, and mating defects. These findings suggest that V-ATPase is essential for endocytosis, ion and pH homeostasis, and for intracellular targeting of vacuolar proteins and vacuolar biogenesis in S. pombe.Communicated by M. Johnston     

The ionic basis of the resting potential of molluscan unstriated muscle

Summary The membrane potential (Vm) of unstriated, non-spiking fibres from the buccal retractor muscle of the opisthobranch molluscPhiline aperta is primarily determined by the distribution of the potassium ion across the membrane. In salines where potassium is varied and chloride remains constant or nearly so, the membrane potential varied with log external K⁺ with a slope of 50.6 (±2.3) mV per decade. In chloride-free salines the slope was 48.5 mV per decade. The experiments were conducted at temperatures of 18–20° C.A ten-fold reduction in external chloride concentration depolarised the fibres by around 10 mV, indicating that chloride permeability makes some contribution to Vm. In salines where [K]₀·[Cl]₀ is constant the Nernst slope was 55.8 mV per decade compared with the theoretical value of 58 mV.The experimental data suggest that the internal potassium concentration of the fibres is 247±31 mM and pNa/pK is 0.01, giving a predicted value of Vm in sea water of –72 mV. The membrane potential of 90 fibres measured in sea water was –74.2±1.3 mV. The membrane contains an electrogenic sodium pump which contributes 4–5 mV to the membrane potential.     

The effect of light on membrane potential, apoplastic pH and cell expansion in leaves of Pisum sativum L. var. Argenteum.

The connection between three light responses of green leaf cells-membrane potential (V_m), H⁺ net efflux and growth, was analyzed. Illumination of mesophyll cells in leaves from Argenteum peas caused two rapid responses: (i) a de- and repolarization of V_m and (ii) an alkalinization of the apoplast. The rapid responses were completely eliminated by the photosynthetic inhibitor 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) but not affected by ortho-vanadate, an inhibitor of the plasma membrane (PM) H⁺-ATPase. The rapid changes were followed by a set of delayed responses: (i) a slow, gradual hyperpolarization of V_m, (ii) a gradual acidification of the mesophyll apoplast and (iii) an increased rate of elongation. These three light responses persisted under DCMU but were completely eliminated by vanadate. The data show that the delayed (in contrast to the rapid) responses were due to a stimulation of PM H⁺ pumps which occurred independently of non-cyclic photosynthetic electron transport and the “dark” processes depending on it. When the rapid responses were blocked by DCMU, light-induced acidification, hyperpolarization of the membrane potential and growth proceeded simultaneously. A shared (4-min) lag phase indicated slower signal processing in mesophyll than in epidermal cells where light stimulation of PM H⁺ pumps was rapid. Received: 3 September 1998 / Accepted: 15 October 1998     

Scanning N-glycosylation mutagenesis of membrane proteins

N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc₃Man₉GlcNAc₂-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12–14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.     

Structure and function of LCI1: a plasma membrane CO2 channel in the Chlamydomonas CO2 concentrating mechanism

Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO₂ in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO₂ concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (C_i) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organism Chlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active C_i uptake systems. Fundamental to eukaryotic C_i uptake systems are C_i transporters/channels located in membranes of various cell compartments, which together facilitate the movement of C_i from the environment into the chloroplast, where primary CO₂ assimilation occurs. Two putative plasma membrane C_i transporters, HLA3 and LCI1, are reportedly involved in active C_i uptake. Based on previous studies, HLA3 clearly plays a meaningful role in HCO₃^? transport, but the function of LCI1 has not yet been thoroughly investigated so remains somewhat obscure. Here we report a crystal structure of the full‐length LCI1 membrane protein to reveal LCI1 structural characteristics, as well as in vivo physiological studies in an LCI1 loss‐of‐function mutant to reveal the C_i species preference for LCI1. Together, these new studies demonstrate LCI1 plays an important role in active CO₂ uptake and that LCI1 likely functions as a plasma membrane CO₂ channel, possibly a gated channel.     

Analyses of the cag pathogenicity island of Helicobacter pylori

Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a ≈37 kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA⁺ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left ‘cagII’ half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis (‘Ptl’), Agrobacterium tumefaciens (‘Vir’), and conjugative plasmids (‘Tra’) that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HslU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end-points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag-encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.