Purified Penicillin Binding Proteins 1Bs from Escherichia coli Membrane Showing Activities of Both Peptidoglycan Polymerase and Peptidoglycan Crosslinking Enzyme

Our biotransformation using Escherichia coli expressing a cytochrome P450 (CYP) belonging to the CYP153A family from Acinetobacter sp. OC4 produced a great amount of 1-octanol (2,250 mg per liter) from n-octane after 24 h of incubation. This level of production is equivalent to the maximum level previously achieved in biotransformation experiments of alkanes. In addition, the initial production rate of 1-octanol was maintained throughout the entire incubation period. These results indicate that we have achieved the functional and stable expression of a CYP in E. coli for the first time. Further, our biotransformation system showed α,ω-diterminal oxidation activity of n-alkanes, and a large amount of 1,8-octanediol (722 mg per liter) was produced from 1-octanol after 24 h of incubation. This is the first report on the bioproduction of α,ω-alkanediols from n-alkanes or 1-alkanols.     

Mutants of Escherichia coli Lacking Endonuclease I, Ribonuclease I, or Ribonuclease II

To isolate mutants of Escherichia coli K-12 lacking endonuclease I activity (end), a method has been developed which detects, by differential methyl green staining, undegraded deoxyribonucleic acid (DNA) in colonies previously incubated in toluene. This procedure allows isolation of mutant strains in which DNA degradation is reduced. For half of these strains, this defect has been correlated with deficiencies of endonuclease I, ribonuclease I (rns), or ribonuclease II (rne) activities. The enzymatic deficiencies of the other strains remain unknown. An rne mutation is cotransducible with serA (which is located at 56 min on the genetic map). Most end mutations, called endA, are also cotransducible with serA and are located between serA and strA. One end mutation, called endB, is located between purE and trp (i.e., between 13 and 25 min on the genetic map).     

Cell Wall Peptidoglycan Mutants of Escherichia coli K-12: Existence of Two Clusters of Genes, mra and mrb, for Cell Wall Peptidoglycan Biosynthesis

Temperature-sensitive mutants of Escherichia coli K-12 which cannot form cell wall peptidoglycan at 42 C were isolated. The thermosensitive steps were characterized biochemically, and the genes coding the enzymes of peptidoglycan synthesis were mapped. These genes were in two clusters: one group, located at about 1.5 min between leu and azi, was designated as mra (murein a); the second group, located at about 77.5 min close to argH and metB, was designated as mrb (murein b, with the order metB-argH-mrb). No simple relations were found between the gene location and the order or localization of enzymes involved in the sequence of reactions of cell wall peptidoglycan biosynthesis.     

In Vitro Peptidoglycan Synthesis Which is Very Sensitive to Cephalexin and Penicillin G in Escherichia coli: Presumable Septum-forming Reaction Sequence

Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SER. var. thunbergii MAKINO, suppressed D-galactosamine-induced liver injury by 85.2% when added to the diet at 1% and fed to rats for fifteen days. The hepatoprotective effect is more potent than that of a milk thistle extract and turmeric powder. Some fractionated extracts showed hepatoprotective activity in the D-galactosamine-induced in vitro liver injury model.     

Eliminating a Set of Four Penicillin Binding Proteins Triggers the Rcs Phosphorelay and Cpx Stress Responses in Escherichia coli

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine. However, the migration defect was caused by the absence of flagella instead of improper flagellar assembly. Migration was restored if the flhDC genes were overexpressed or if the rcsB or cpxR genes were deleted. Thus, migration was inhibited because the Rcs and Cpx stress response systems were induced in the absence of these four specific PBPs. Furthermore, in this situation Rcs induction depended on the presence of CpxR. The results imply that small changes in peptidoglycan structure are sufficient to activate these stress responses, suggesting that a specific cell wall fragment may be the signal being sensed. The fact that four PBPs must be inactivated may explain why large perturbations to the envelope are required to induce stress responses.     

Genetic and Molecular Characteristics of X-Ray-Sensitive Mutants of Escherichia coli Defective in Repair Synthesis

Alleles responsible for X-ray-sensitive characteristics of three mutants of Escherichia coli B, which were also sensitive to ultraviolet (UV) irradiation, were mapped near metE locus, and named res-1, res-2, and res-3. All the res(-) mutants showed no host cell reactivability (Hcr(-)) for transducing deoxyribonucleic acid (DNA) of P1 phage irradiated by UV but they were Hcr(+) for infective DNA of P1 phage. Furthermore, they showed no detectable activity of DNA polymerase. Characteristics of allele res-1 were studied in detail. The mutant res-1 uvr(+) showed an extensive degradation of DNA after UV irradiation. Double mutants carrying res-1 uvrA(-), res-1 uvrB(-), and res-1 uvrC(-) showed no marked increase in UV sensitivity beyond that of the uvr(-) single mutants and only negligible UV-induced DNA degradation. The uvr(-) mutations showed no such suppressive effect on DNA degradation induced by X rays in these double mutants. It is concluded that res(-) mutants are defective in the second step (repair synthesis) of the excision repair process and that DNA polymerase is partly responsible for the assumed resynthesis step.     

Localization and Function of Early Cell Division Proteins in Filamentous Escherichia coli Cells Lacking Phosphatidylethanolamine

Escherichia coli cells that contain the pss-93 null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE). Such cells are defective in cell division. To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected. Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in pss-93 filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE. However, there was no evidence of constriction at most of these potential division sites. FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments. This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP. The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction.     

Growth of Escherichia coli on Short-Chain Fatty Acids: Growth Characteristics of Mutants

The parent Escherichia coli K-12 is constitutive for the enzymes of the glyoxylate bypass and adapts to growth on long-chain fatty acids (C(12) to C(18)). It does not utilize medium-chain (C(6) to C(11)) or short-chain (C(4), C(5)) n-monocarboxylic acids. Several mutants of this strain which grow using short- or medium-chain acids, or both, as the sole carbon source were selected and characterized. One mutant (D(1)) synthesizes the beta-oxidation enzymes constitutively and grows on medium-chain but not on short-chain acids. A second (N(3)) is partially derepressed for synthesis of these enzymes and grows both on medium-chain and on short-chain acids. Secondary mutants (N(3)V(-), N(3)B(-), N(3)OL(-)) were derived from N(3). N(3)V(-) grows on even-chain but not on odd-chain acids and exhibits a lesion in propionate oxidation. N(3)B(-) grows on odd-chain but not on even-chain acids and exhibits no crotonase activity as assayed by hydration of crotonyl-CoA. N(3)OL(-) grows on acetate and propionate but does not utilize fatty acids C(4) to C(18); it exhibits multiple deficiencies in the beta-oxidation pathway. Growth on acetate of N(3), but not of the parent strain, is inhibited by 4-pentenoate. Revertants of N(3) which are resistant to growth inhibition by 4-pentenoate (N(3)PR) exhibit loss of ability to grow on short-chain acids but retain the ability to grow on medium-chain and long-chain acids. The growth characteristics of these mutants suggest that in order to grow at the expense of butyrate and valerate, E. coli must be (i) derepressed for synthesis of the beta-oxidation enzymes and (ii) derepressed for synthesis of a short-chain fatty acid uptake system.     

大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用

大肠杆菌分泌蛋白二硫键的形成是一系列蛋白协同作用的结果,主要是Dsb家族蛋白,迄今为止共发现了DsbA、DsbB、DsbC、DsbD、DsbE和DsbG。在体内,DsbA负责氧化两个巯基形成二硫键,DsbB则负责DsbA的再氧化。DsbC和DsbG负责校正DsbA导入的异常二硫键,DsbD则负责对DsbC和DsbG进行再还原,DsbE的功能与DsbD类似。除了直接和二硫键的形成相关外,DsbA、DsbC和DsbG都有分子伴侣功能。它们的分子伴侣功能独立于二硫键形成酶的活性并且对二硫键形成酶活性具有明显的促进作用。基于Dsb蛋白的功能特性,利用它们以大肠杆菌为宿主表达外源蛋白,特别是含有二硫键的蛋白,取得了很多成功的例子。本文简要介绍了这方面的进展,显示Dsb蛋白在促进外源蛋白在大肠杆菌中以可溶形式表达方面具有广阔的应用前景。     

Synthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C

In response to the widespread use of β-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent β-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3 μM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50) = 26 μM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50) = 138 μM) and a class C PBP (R39 from Actinomadura sp., IC(50) = 0.6 μM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species.     

Mutants of Escherichia coli Defective in Membrane Phospholipid Synthesis: Mapping of sn-Glycerol 3-Phosphate Acyltransferase Km Mutants

plsB mutants of Escherichia coli are sn-glycerol 3-phosphate auxotrophs which owe their requirement to a K(m) defect in sn-glycerol 3-phosphate acyltransferase, the first enzyme in the phospholipid biosynthetic pathway. We have located the plsB gene at minute 69 of the E. coli genetic map, far removed from the gene defined by mutants with a temperature-sensitive sn-glycerol 3-phosphate acyltransferase. The plsB gene was cotransduced with the dctA locus, and the transduction data indicated that the clockwise gene order is asd, plsB, dctA, xyl. plsB(-) is recessive to plsB(+) and all acyltransferase K(m) mutants tested lie very close to the plsB locus. Effective supplementation of plsB mutants was shown not to require a defective glpD gene.     

De Novo Biosynthesis of Nicotinamide Adenine Dinucleotide in Escherichia coli: Excretion of Quinolinic Acid by Mutants Lacking Quinolinate Phosphoribosyl Transferase

The excretion of quinolinic acid was studied in growing and resting cells of Escherichia coli K-12 nadC(13). Under optimal conditions, this organism could synthesize quinolinic acid in several-fold excess of the amount which would be required for normal growth. The excretion of quinolinic acid was controlled by the concentration of nicotinamide adenine dinucleotide (NAD) precursors available to the organism either during growth or during incubation in dense cell suspensions. These observations suggest that biosynthesis of NAD de novo is regulated by both repression and feedback inhibition. Analogues of niacin which inhibit bacterial growth also inhibited and repressed the synthesis (excretion) of quinolinic acid. The pH optimum for quinolinic acid excretion agreed favorably with the optimum observed for its synthesis in vitro. The rate of quinolinic acid excretion was strongly influenced by the concentration of ribose or glycerol in the medium.     

Manganese-Resistant Mutants of Escherichia coli: Physiological and Genetic Studies

Manganese is growth inhibitory for Escherichia coli. The manganese concentration required for inhibition is dependent upon the magnesium concentration of the medium. Mutants have been isolated which are partially resistant to manganese inhibition in both liquid and solid media. From conjugation experiments, the genetic locus for manganese-resistance, mng, appears to be between 34 and 37 min on the E. coli genetic map. Experiments with radioactive (28)Mg lead to the tentative conclusion that the mng mutants are altered in the inhibition constant for manganese as a competitive inhibitor for the mangnesium accumulation system. Once high manganese enters the cells, it displaces internal magnesium and leads to a net cellular loss and hence growth inhibition. The mng mutants are somewhat less subject to manganese-induced magnesium loss under comparable conditions than are manganese-sensitive wild-type cells.     

Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: MINIMAL LIPOPOLYSACCHARIDE STRUCTURE AND INDUCTION OF ENVELOPE STRESS RESPONSE*

To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Δ(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 °C, besides showing accumulation of free lipid IV_A precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Δ(waaC lpxL lpxM lpxP) strains showed primarily Kdo₂-lipid IV_A, indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 °C. These lipid IV_A derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. ΔwaaA and Δ(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and σ^E signal transduction pathways. Δ(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Δ(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Δ(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II.Lipopolysacharides (LPS)4 are the major amphiphilic constituents of the outer leaflet of the outer membrane (OM) of Gram-negative bacteria, including Escherichia coli. LPS share a common architecture composed of a membrane-anchored phosphorylated and acylated β(1→6)-linked GlcN disaccharide, termed lipid A, to which a carbohydrate moiety of varying size is attached (1, 2). The latter may be divided into a lipid A proximal core oligosaccharide and, in smooth-type bacteria, a distal O-antigen. LPS always contain 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo) linked to the lipid A.The physiological importance of the Kdo/lipid A region is reflected by its specific position within the pathway of LPS biosynthesis. In E. coli K-12, a bisphosphorylated lipid A precursor molecule with two amide and two ester-bound (R)-3-hydroxymyristate residues (lipid IV_A) is synthesized from UDP-GlcNAc, following 6 distinct enzyme reactions (1). This intermediate serves as an acceptor for the Kdo transferase (WaaA), which transfers two Kdo residues from CMP-Kdo to yield an α(2→4)-linked Kdo disaccharide-attached α(2→6) to the non-reducing GlcN residue of lipid IV_A (3). The latter reaction product, termed Kdo₂-lipid IV_A, comprises a key intermediate of LPS biosynthesis that acts 2-fold as a specific substrate: (i) for glycosyltransferases catalyzing further steps of the core oligosaccharide biosynthesis (4) and (ii) for acyltransferases that complete the lipid A moiety by the transfer of 2 additional fatty acids to the (R)-3-hydroxyl groups of both acyl chains, which are directly bound to position 2′ and 3′ of the non-reducing GlcN residue (1). Three acyltransferases, encoded by paralogous genes, have been described in E. coli K-12, which catalyze the latter enzyme reactions using acyl carrier protein-activated fatty acids as co-substrates (5–10). At ambient temperatures, a lauroyl residue is first transferred by LpxL (6) to the OH group of the amide-bound (R)-3-hydroxymyristate residue at position 2′. This catalytic step is partially replaced at low temperature (12 °C) by LpxP, which transfers palmitoleate to the same position in ∼80% of the LPS molecules (7). The free OH group of the ester-bound (R)-3-hydroxymyristate residue at position 3′ within both pentaacylated intermediates is then myristoylated by LpxM to give a hexaacylated lipid A moiety (Fig. 3) (5).Open in a separate windowFIGURE 3.Chemical structure of tetraacylated lipid IV_A precursor (A) and Kdo₂-lipid IV_A (B). R1 represents C12:0 or C16:1; R2, C14:0; R3 and R4 are under LPS-modifying conditions P-EtN and l-Ara4N, respectively, and R5, C16:0.Consistent with the essentiality of LPS in E. coli, all the genes, whose products are required for committed steps of biosynthesis of lipid IV_A and subsequent transfer of Kdo to it, are essential (1, 2). However, individually neither the subsequent steps of addition of the secondary lauroyl and myristoyl residues to the distal glucosoamine unit by LpxL and LpxM to synthesize hexaacylated lipid A nor the later glycosylation of hexaacylated Kdo₂-lipid A is essential for viability of bacteria like E. coli K-12 under defined growth conditions (8). Although Re mutants that possess LPS with only hexaacylated Kdo₂-lipid A or mutants that synthesize complete LPS core with only lipid IV_A are viable, they are impaired in several growth properties, including constitutive induction of RpoE signal transduction in Re mutants (8, 11–13). A triple null mutant, which lacks all 3 late acyltransferases, is viable but only in slow-growth conditions in accordance with lipid IV_A being a poor substrate of the lipid A transporter MsbA (8). Mutants impaired in the synthesis of Kdo, which synthesize only lipid IV_A lacking any glycosylation, can be constructed, but they require additional suppressor mutations either in msbA, or the yhjD gene (14, 15). Strains that potentially can only synthesize Kdo₂-lipid IV_A have not been reported up to now. Thus, suppressor-free minimal LPS structures that can support growth of E. coli K-12 bacteria known up to now have genetic compositions of Δ(lpxL lpxM lpxP) or Re mutants.We describe the construction and characterization of suppressor-free ΔwaaA and Δ(waaC lpxL lpxM lpxP) mutants, synthesizing either free lipid IV_A derivatives or Kdo₂-lipid IV_A LPS, respectively. Analyses of lipid A of ΔwaaA also revealed the presence of free penta- and hexaacylated lipid A derivatives, arising due to incorporation of secondary acyl chains. Such suppressor-free strains could be constructed only in slow-growth conditions at lower temperatures. Growth of Δ(waaC lpxL lpxM lpxP) could be restored by extragenic chromosomal MsbA-D498V suppressor mutation or by the overexpression of the msbA wild-type gene product. The LPS of Δ(waaC lpxL lpxM lpxP) and lipid IV_A precursor of ΔwaaA was found to be substituted by P-EtN, but not l-Ara4N, under LPS-modifying growth conditions. Deletion of late acyltransferases in ΔwaaC or deletion of the waaA gene resulted in constitutively elevated levels of periplasmic protease HtrA, due to additive induction of the envelope stress responsive CpxR/A two-component system and σ^E pathway.     

Murein Synthesis and Identification of Cell Wall Precursors of Temperature-Sensitive Lysis Mutants of Escherichia coli

A group of temperature-sensitive lysis mutants of Escherichia coli K-12 was studied. Mutants impaired in the synthesis of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc)-pentapeptide or in the synthesis of murein amino acids were found. Their rate of murein synthesis at the restrictive temperature was decreased. A large number of mutants did not differ from the parent strain with respect to the rate of murein synthesis and the precursor pattern. The behavior of these mutants is discussed. It was impossible to accumulate UDP-MurNAc-pentapeptide in E. coli by the antibiotics penicillin and vancomycin. The hypothesis is put forward that the amount of this murein precursor is regulated by feedback inhibition.     

Comparative Proteomic Analysis of Extracellular Proteins of Enterohemorrhagic and Enteropathogenic Escherichia coli Strains and Their ihf and ler Mutants

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.     

Effect of Dichlorodifluoromethane on the Appearance, Viability, and Integrity of Escherichia coli

Cultures of Escherichia coli H52 were treated with liquid dichlorodifluoromethane (fluorocarbon-12 [f-12]) for 2 h at 22 C and then examined microscopically. Treated cells tended to clump, and their cytoplasms were generally less dense and less uniform in appearance than those of control cells. E. coli ML30 was exposed to f-12 at a concentration of 1.25 × saturation for times up to 1,200 min at 22 C. Cells were examined for changes in viability (plate count), permeability (as measured by exit of α-[¹⁴C]methylglucoside or uptake of o-nitrophenyl-β-D-galactopyranoside), release of compounds absorbing at 260 nm, and lysis (changes in absorbance at 420 nm). Large losses of α-methylglucoside and of percentage of viability occurred after brief exposure to f-12. Release of compounds absorbing at 260 nm occurred more slowly than the aforementioned events, possibly because these molecules are larger than α-methylglucoside. During 1,200-min exposure to f-12, the number of survivors decreased from 10⁹ to 10⁴ organisms/ml, the loss of compounds absorbing at 260 nm amounted to 50% and 32% lysis occurred. Most of these changes occurred during the first 300 min of treatment. Loss of α-methylglucoside was almost complete after 1-min exposure to f-12. These results suggest that death of the cell involves several stages, with a change of permeability occurring first, followed by leakage of compounds of increasing size and, finally, lysis.