AppppA-binding protein E89 is the Escherichia coli heat shock protein ClpB.

Dinucleotide AppppA (5',5''-P1,P4-diadenosine tetraphosphate) is rapidly synthesized in Escherichia coli cells during heat shock. apaH mutants lack AppppN hydrolase activity and, therefore, contain constitutively levels of AppppA, which affect several cellular processes. However, the precise role of AppppA remains undetermined. Photo-crosslinking experiments with radioactively labelled azido-AppppA have shown that a number of proteins, including heat shock proteins DnaK and GroEL, specifically bind to AppppA. Several other unidentified proteins (C40, C45, and E89) also bind strongly to AppppA. In this work, we have identified the AppppA-binding protein E89 as heat shock protein ClpB. In addition, since ClpB belongs to a family of proteins implicated in proteolysis, we have examined the effects of apaH mutants on protein degradation. Constitutively elevated levels of AppppA stimulate lon-independent proteolysis only in heat-shocked cells. We also show that overproduction of ClpB from a plasmid rescues apaH mutants from sensitivity to killing by heat.     

HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise

Exercise causes heat shock (muscle temperatures of up to 45 degrees C, core temperatures of up to 44 degrees C) and oxidative stress (generation of O2- and H2O2), and exercise training promotes mitochondrial biogenesis (2-3-fold increases in muscle mitochondria). The concentrations of at least 15 possible heat shock or oxidative stress proteins (including one with a molecular weight of 70 kDa) were increased, in skeletal muscle, heart, and liver, by exercise. Soleus, plantaris, and extensor digitorum longus (EDL) muscles exhibited differential protein synthetic responses ([3H]leucine incorporation) to heat shock and oxidative stress in vitro but five proteins (particularly a 70 kDa protein and a 106 kDa protein) were common to both stresses. HSP70 mRNA levels were next analyzed by Northern transfer, using a [32P]-labeled HSP70 cDNA probe. HSP70 mRNA levels were increased, in skeletal and cardiac muscle, by exercise and by both heat shock and oxidative stress. Skeletal muscle HSP70 mRNA levels peaked 30-60 min following exercise, and appeared to decline slowly towards control levels by 6 h postexercise. Two distinct HSP70 mRNA species were observed in cardiac muscle; a 2.3 kb mRNA which returned to control levels within 2-3 h postexercise, and a 3.5 kb mRNA species which remained at elevated concentrations for some 6 h postexercise. The induction of HSP70 appears to be a physiological response to the heat shock and oxidative stress of exercise. Exercise hyperthermia may actually cause oxidative stress since we also found that muscle mitochondria undergo progressive uncoupling and increased O2- generation with increasing temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of oxidative stress on homer scaffolding proteins

Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.     

Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants.

To examine functions of two small heat shock proteins of Escherichia coli, IbpA and IbpB, we constructed His-IbpA and His-IbpB, in which a polyhistidine tag was fused to the N-terminals. Both purified His-IbpA and His-IbpB formed multimers, which have molecular masses of about 2.0-3.0 MDa and consist of about 100-150 subunits. They suppressed the inactivation of several enzymes including citrate synthase and 6-phosphogluconate dehydrogenase by heat, potassium superoxide, hydrogen peroxide and freeze-thawing, but not the inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide. Both His-IbpA and His-IbpB suppressed enzyme inactivation by various treatments and were also found to be associated with their non-native forms. However, both His-IbpA and His-IbpB were not able to reactivate enzymes inactivated by heat, oxidants or guanidine hydrochloride. When heated to 50 degrees C, each multimeric form of His-IbpA or His-IbpB was dissociated to form a monomer for His-IbpA, and an oligomer of about one-quarter size for His-IbpB. These structural changes were reversible, as both heated proteins regained the multimeric structures after incubation at 25 degrees C. However, when exposed to hydrogen peroxide or potassium superoxide, the large multimeric forms of His-IbpA and His-IbpB were maintained. The results suggest that His-IbpA and His-IbpB suppress the inactivation of enzymes and bind non-native proteins to protect their structures from heat and oxidants.     

Proteomic analysis of heat-stable proteins in Escherichia coli

Some proteins of E. coli are stable at temperatures significantly higher than 49 degrees C, the maximum temperature at which the organism can grow. The heat stability of such proteins would be a property which is inherent to their structures, or it might be acquired by evolution for their specialized functions. In this study, we describe the identification of 17 heat-stable proteins from E. coli. Approximately one-third of these proteins were recognized as having functions in the protection of other proteins against denaturation. These included chaperonin (GroEL and GroES), molecular chaperones (DnaK and FkpA) and peptidyl prolyl isomerases (trigger factor and FkpA). Another common feature was that five of these proteins (GroEL, GroES, Ahpc, RibH and ferritin) have been shown to form a macromolecular structure. These results indicated that the heat stability of certain proteins may have evolved for their specialized functions, allowing them to cope with harsh environments, including high temperatures.     

Degradation of oxidatively denatured proteins in Escherichia coli

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein synthesis inhibitors and ethanol selectively enhance heterologous expression of P450s and related proteins in Escherichia coli.

The antibiotics chloramphenicol (Cm), tetracycline, and erythromycin, which inhibit bacterial protein synthesis and are known to induce the cold shock response, unexpectedly enhance the heterologous expression of P450s and related proteins in Escherichia coli. In contrast, antibiotics that mimic heat shock in E. coli such as puromycin, streptomycin, and kanamycin decrease the expression of the same proteins. A sublethal dose of Cm (1 microgram/ml) effectively enhances the expression of both membrane-bound proteins (microsomal and mitochondrial P450s) and a soluble mitochondrial protein (adrenodoxin) over the range of two- to eightfold. The expression level of N-terminal truncated P450c17 (1600 nmol/liter culture without Cm), for instance, reached 3500 nmol/liter culture by the addition of Cm, approximately 8.4% of the total cellular protein. Cm also enabled expression at useful levels of active P450s previously difficult to express in E. coli. In contrast, the expression of P450scc, a mitochondrial protein, is decreased by Cm but enhanced by ethanol, a powerful elicitor of heat shock response in E. coli. These results show that both the cold shock response induced by some antibiotics and the heat shock response induced by ethanol may lead to enhanced expression of certain heterologous proteins in E. coli. This study also indicates that protein synthesis inhibitors associated with the cold shock response may act as protein synthesis enhancers under certain conditions.     

Crucial role of antioxidant proteins and hydrolytic enzymes in pathogenicity of Penicillium expansum: analysis based on proteomics approach

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase.     

High resistance to oxidative damage in the Antarctic midge Belgica antarctica, and developmentally linked expression of genes encoding superoxide dismutase, catalase and heat shock proteins

Intense ultraviolet radiation, coupled with frequent bouts of freezing-thawing and anoxia, have the potential to generate high levels of oxidative stress in Antarctic organisms. In this study, we examined mechanisms used by the Antarctic midge, Belgica antarctica, to counter oxidative stress. We cloned genes encoding two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), and showed that SOD mRNA was expressed continuously and at very high levels in larvae, but not in adults, while Cat mRNA was expressed in both larvae and adults but at a somewhat reduced level. SOD mRNA was expressed at even higher levels in larvae that were exposed to direct sunlight. Catalase, a small heat shock protein, Hsp70 and Hsp90 mRNAs were also strongly upregulated in response to sunlight. Total antioxidant capacity of the adults was higher than that of the larvae, but levels in both stages of the midge were much higher than observed in a freeze-tolerant, temperate zone insect, the gall fly Eurosta solidaginis. Assays to measure oxidative damage (lipid peroxidation TBARS and carbonyl proteins) demonstrated that the Antarctic midge is highly resistant to oxidative stress.     

A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana

Among the heat shock proteins (HSPs) of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. To improve our understanding of sHSPs, we have characterized RcHSP17.8 from Rosa chinensis . Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. RcHSP17.8 expression in R. chinensis was induced by heat, cold, salt, drought, osmotic and oxidative stresses. Recombinant RcHSP17.8 was overexpressed in Escherichia coli and yeast to study its possible function under stress conditions. The recombinant E. coli and yeast cells that accumulated RcHSP17.8 showed improved viability under thermal, salt and oxidative stress conditions compared with control cultures. We also produced transgenic Arabidopsis thaliana that constitutively expressed RcHSP17.8. These plants exhibited increased tolerance to heat, salt, osmotic and drought stresses. These results suggest that R. chinensis cytosolic class I sHSP (RcHSP17.8) has the ability to confer stress resistance not only to E. coli and yeast but also to plants grown under a wide variety of unfavorable environmental conditions.     

Identification of proteins of Escherichia coli and Saccharomyces cerevisiae that specifically bind to C/C mismatches in DNA

The pathways leading to G:C→C:G transversions and their repair mechanisms remain uncertain. C/C and G/G mismatches arising during DNA replication are a potential source of G:C→C:G transversions. The Escherichia coli mutHLS mismatch repair pathway efficiently corrects G/G mismatches, whereas C/C mismatches are a poor substrate. Escherichia coli must have a more specific repair pathway to correct C/C mismatches. In this study, we performed gel-shift assays to identify C/C mismatch-binding proteins in cell extracts of E.coli. By testing heteroduplex DNA (34mers) containing C/C mismatches, two specific band shifts were generated in the gels. The band shifts were due to mismatch-specific binding of proteins present in the extracts. Cell extracts of a mutant strain defective in MutM protein did not produce a low-mobility complex. Purified MutM protein bound efficiently to the C/C mismatch-containing heteroduplex to produce the low-mobility complex. The second protein, which produced a high-mobility complex with the C/C mismatches, was purified to homogeneity, and the amino acid sequence revealed that this protein was the FabA protein of E.coli. The high-mobility complex was not formed in cell extracts of a fabA mutant. From these results it is possible that MutM and FabA proteins are components of repair pathways for C/C mismatches in E.coli. Furthermore, we found that Saccharomyces cerevisiae OGG1 protein, a functional homolog of E.coli MutM protein, could specifically bind to the C/C mismatches in DNA.     

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.     

Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima.

An expressed sequence tag homologous to cheA was previously isolated by random sequencing of Thermotoga maritima cDNA clones (C. W. Kim, P. Markiewicz, J. J. Lee, C. F. Schierle, and J. H. Miller, J. Mol. Biol. 231: 960-981, 1993). Oligonucleotides complementary to this sequence tag were synthesized and used to identify a clone from a T. maritima lambda library by using PCR. Two partially overlapping restriction fragments were subcloned from the lambda clone and sequenced. The resulting 5,251-bp sequence contained five open reading frames, including cheA, cheW, and cheY. In addition to the chemotaxis genes, the fragment also encodes a putative protein isoaspartyl methyltransferase and an open reading frame of unknown function. Both the cheW and cheY genes were individually cloned into inducible Escherichia coli expression vectors. Upon induction, both proteins were synthesized at high levels. T. maritima CheW and CheY were both soluble and were easily purified from the bulk of the endogenous E. coli protein by heat treatment at 80 degrees C for 10 min. CheY prepared in this way was shown to be active by the demonstration of Mg(2+)-dependent autophosphorylation with [32P]acetyl phosphate. In E. coli, CheW mediates the physical coupling of the receptors to the kinase CheA. The availability of a thermostable homolog of CheW opens the possibility of structural characterization of this small coupling protein, which is among the least well characterized proteins in the bacterial chemotaxis signal transduction pathway.     

Heat shock causes oxidative stress and induces a variety of cell rescue proteins in Saccharomyces cerevisiae KNU5377

In this study, we attempted to characterize the physiological response to oxidative stress by heat shock in Saccharomyces cerevisiae KNU5377 (KNU5377) that ferments at a temperature of 40 degrees C. The KNU5377 strain evidenced a very similar growth rate at 40 degrees C as was recorded under normal conditions. Unlike the laboratory strains of S. cerevisiae, the cell viability of KNU5377 was affected slightly under 2 hours of heat stress conditions at 43 degrees C. KNU5377 evidenced a time-dependent increase in hydroperoxide levels, carbonyl contents, and malondialdehyde (MDA), which increased in the expression of a variety of cell rescue proteins containing Hsp104p, Ssap, Hsp30p, Sod1p, catalase, glutathione reductase, G6PDH, thioredoxin, thioredoxin peroxidase (Tsa1p), Adhp, Aldp, trehalose and glycogen at high temperature. Pma1/2p, Hsp90p and H+-ATPase expression levels were reduced as the result of exposure to heat shock. With regard to cellular fatty acid composition, levels of unsaturated fatty acids (USFAs) were increased significantly at high temperatures (43 degrees C), and this was particularly true of oleic acid (C18:1). The results of this study indicated that oxidative stress as the result of heat shock may induce a more profound stimulation of trehalose, antioxidant enzymes, and heat shock proteins, as well as an increase in the USFAs ratios. This might contribute to cellular protective functions for the maintenance of cellular homeostasis, and may also contribute to membrane fluidity.     

Molecular and biochemical modulation of heat shock protein 20 (Hsp20) gene by temperature stress and hydrogen peroxide (H₂O₂) in the monogonont rotifer, Brachionus sp

The monogonont rotifer, Brachionus sp. has been regarded as a potential model for reproductive physiology, evolution, and environmental genomics. To uncover the role of the heat shock protein upon temperature stress and hydrogen peroxide (H?O?) exposure, we cloned heat shock protein 20 (Hsp20) and determined its modulatory response under different temperatures and H?O? concentrations. Under different temperature stresses (10 °C and 37 °C), the rotifer Brachionus sp. Hsp20 (Br-Hsp20) gene was highly expressed over time, and reached the maximum level 90 min after exposure, indicating that Br-Hsp20 gene would be involved in the chaperoning process to protect proteins at both low and high temperatures. To test the ability of thermotolerance of the recombinant Br-Hsp20-containing transformed Escherichia coli, we expressed the recombinant Br-Hsp20 protein with 1mM IPTG for 18 h at 30 °C, exposed them at 54 °C with time course (10 to 60 min), and measured cell survival. In this elevated temperature shock (54 °C), the cell survival was significantly higher at the Br-Hsp20 transformed E. coli, compared to the control (vector only). To analyze the modulatory effect of Br-Hsp20 gene on oxidative stress, we initially exposed 0.1 mM H?O? over time and measured antioxidant enzyme activities along with the expression level of Br-Hsp20 mRNA. Upon H?O? exposure, Br-Hsp20 gene was time-dependently upregulated and glutathione peroxidase (GPx), glutathione S-transferase (GST), and glutathione reductase (GR) activities were also elevated at the 12h-exposed group in a dose-dependent manner, indicating that the Br-Hsp20 gene would be an important gene in response to oxidative and temperature stress. Here, we demonstrated the role of the Hsp20 gene in the rotifer, Brachionus sp. providing a better understanding of the ecophysiology at environmental stress in this species.     

Escherichia coli DnaK protein possesses a 5''-nucleotidase activity that is inhibited by AppppA.

AppppA and the DnaK protein have both been hypothesized to function in regulating the heat shock response of Escherichia coli. The proposals are that AppppA serves as a signal (alarmone) to turn on the heat shock response, whereas the DnaK protein is necessary to turn off the heat shock response. A simple model would be that the DnaK protein turns off the response by degrading AppppA. We disproved this model by demonstrating that the DnaK protein possesses a 5'-nucleotidase activity capable of degrading many cellular nucleotides but not AppppA. Although AppppA was not a substrate, it did inhibit the 5'-nucleotidase activity of the DnaK protein. This inhibition may be specific and have biological function since the mutant DnaK756 protein, which is defective in turning off the heat shock response, is partially desensitized to AppppA inhibition. These findings led us to consider other possible mechanisms for AppppA and the DnaK protein in heat shock regulation.     

Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli

To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain. The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress. However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain. The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB. These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress.     

Thermoinduced radioresistance of Escherichia coli cells and heat shock proteins

Radioresistance of E. coli cells is slightly increased (dose modification factor (DMF) = 1.2) with temperature elevated from 4 degrees to 43 degrees C at the time of gamma-irradiation. However, an appreciable effect of the thermoinduced radioresistance (DMF = 1.7) was observed when the wild-type cells were exposed to gamma-radiation at 15-43 degrees C (but not at 4 degrees C) after 30-min preincubation at 43 degrees C. This effect was absent in htpR mutants, defective in induction of heat shock proteins, and coupled with the decreased post-irradiation DNA degradation in gamma-irradiated htpR+ cells. It is suggested that heat shock proteins are involved in the thermoinduced radioresistance.     

Comparative studies of penicillin-binding proteins in Pseudomonas aeruginosa and Escherichia coli.

Penicillin-binding proteins in Pseudomonas aeruginosa were compared with those of Escherichia coli. These in P. aeruginosa were found exclusively in the cytoplasmic membrane fraction (fraction soluble in sodium N-lauroyl sarcosinate). Sodium dodecyl sulfate/acrylamide gel electrophoresis of the proteins bound to [14C]penicillin G resulted in the separation of six major bands and several minor bands. The proteins in these bands are referred to as proteins 1A, 1B, 2, 3, 4 and 5 in order of increasing electrophoretical mobility. The electrophoretic mobilities and other properties of penicillin-binding proteins in P. aeruginosa and E. coli were compared and correlated. Fundamentally they seem to be very similar in the two bacteria, but proteins 1A and 1B in P. aeruginosa seem to correspond respectively to proteins 1B and 1A in E. coli, and protein 6 seems to be missing or present in only small amount in P. aeruginosa. In addition, the affinities of currently developed beta-lactam antibiotics to each protein of P. aeruginosa and E. coli were examined in relation to the morphological changes of the cells induced by these antibiotics and their antibacterial potencies. Mecillinam showed high affinity to only protein 2 in both P. aeruginosa and E. coli. At a minimal inhibitory concentration, it converted cells of both P. aeruginosa and E. coli from rods to spherical cells, although its minimal inhibitory concentration was much higher for P. aeruginosa than for E. coli.