Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.     

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.     

氯吡格雷对大鼠肝药物酶CYP3A4和CYP1A2表达的影响

目的：氯吡格雷主要由CYP3A4催化使其激活,CYPlA2也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4和CYPlA2的表达,探讨氯吡格雷对大鼠肝药物酶的影响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13．5,6．75mg／kg／d）,雄性健康大鼠连续灌胃给药7天,脱臼处死,取肝组织,通过westernblot法检测大鼠肝脏CYP3A4和CYPlA2蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4蛋白表达量降低（P〈0．05）;氯吡格雷低中高剂量组间进行比较,大鼠CYP3A4蛋白表达量呈梯度减少（P〈0．05）;2）、氯吡格雷抑制大鼠CYPlA2蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYPlA2蛋白表达量降低（P〈0．05）,氯吡格雷低中高剂量组间进行比较,大鼠CYPlA2蛋白表达量呈梯度减少（P〈0．05）。结论：氯吡格雷使肝细胞色素氧化酶CYP3A4和CYPlA2的表达量减少,因此氯吡格雷高、中、低3个剂量组均不同程度的抑制大鼠肝脏CYP3A4和CYPlA2的表达,提示当氯吡格雷与某些主要经CYP3A4和CYPlA2代谢的药物合用时,发生代谢性相关作用的可能性大。     

大肠杆菌外膜蛋白的分离及其双向电泳图谱的建立

本文利用温度诱导的两相分离萃取技术选择性分离未经机械破碎的大肠杆菌细胞外膜蛋白,研究TritonX.114的浓度及处理时间对提取外膜蛋白的影响.实验结果表明,Triton X-114的使用浓度和作用时间均显著影响外膜蛋白的提取效率.SDS-PAGE结果表明不同Triton X-114的使用浓度和作用时间只是影响了外膜蛋白的提取效率而对外膜蛋白提取的种类没有影响.实验发现8%的Triton X-114处理3小时为最佳分离条件,分离得到的样品可用于双向电泳分析.通过对比实验发现样品裂解液中包含低浓度的Tris是外膜蛋白双向电泳成功的关键因素,CHAPS与ASB-14或NP-40结合使用可显著提高外膜蛋白的溶解能力,缩短聚焦时间,从而优化了大肠杆茵外膜蛋白双向电泳技术体系,建立了其双向电泳图谱.     

大黄鱼CYP1A基因cDNA的克隆与表达分析

由于外源化合物能诱导鱼类CYPIA(P4501A)的表达,因而它广泛被用作评价水环境污染生物标记物.利用RT-PCR结合RACE技术从大黄鱼(Larimichthys crocea)肝脏克隆了CYP1A基因全长cDNA序列.经分析,该cDNA的5'末端有175 bp的非翻译区.开放阅读框为1 566 bp,编码521个氨基酸和一个终止密码子,3'末端有857 bp的非翻译区,3'非翻译区有一个多聚腺苷酸信号及两个与mRNA的快速降解有关的AUUUA序列.推测大黄鱼CYP1A的氨基酸序列和欧洲鲈鱼的相似度最高迭89.6%.用RT-PCR检测大黄鱼CYP1A的表达特征发现,在所检测的9个组织中均有表达,以肝脏、消化道、脾脏和肾脏的表达量较高.     

Fragmentation of Penicillin-binding Protein-1Bs of Escherichia coli by Proteolytic Enzymes: A Preliminary Study on the Location of the Penicillin-binding Site on the Bifunctional Peptidoglycan Synthetase Protein

The absolute configurations of fenvalerate and other related cyanohydrin esters were studied by circular dichroism (CD) measurements and by high performance liquid chromatography (HPLC). Fenvalerate has UV absorption peaks around 278 nm associated with the ¹L_b phenyl transitions and corresponding positive CD peaks were observed around 281 nm for the enantiomers of (S)-configuration at the cyanohydrin chiral center. Most of the other cyanohydrin esters also gave positive CD peaks for the enantiomers of (S)-configuration. CD spectra in the 180 to 250 nm range were also studied.

By HPLC, the elution order of the diastereoisomers of cyanohydrin esters were closely correlated with their absolute configuration and the (RS,SR)-pair consistently eluted earlier than the (RR,SS)-pair for α-substituted phenylacetic acid esters.     

Purification and Characterization of Cry1Ac Toxin Binding Proteins from the Brush Border Membrane of Helicoverpa armigera Midgut

Several Cry1Ac binding proteins from midgut of Helicoverpa armigera were purified using toxin-affinity chromatography. Enzyme assays showed that the purified proteins had strong aminopeptidase activity. The N-terminal sequences confidently identified a 124-kDa binding protein as an aminopeptidase N (APN), and some similarity suggests that a 162-kDa binding protein may also be an APN. Two minor binding proteins were not characterized.     

Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, Delta 46A1H had a 4x His-tag fused to the C-terminus; H Delta 46A1 had the N-terminal 4x His-tag; H Delta 46A1 Delta had a 4x His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and Delta 46A1 Delta lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, Delta 46A1 Delta and H Delta 46A1 Delta, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.     

Purification and Characterization of DNA Polymerase Obtained from the Membrane Fraction of E. coli

Purification studies were conducted on DNA polymerase bound to the membrane fraction of E. coli HF 4704. Purified enzyme (Fraction V) required Mg²⁺ and showed an optimun pH of 7.2. Various kinds of salt indicated a stimulative effect at concentrations lower than 0.1 m. Fraction V was unstable at an acidic condition (pH 5.0) but was rather stable at an alkaline condition (pH 9.0). The enzyme activity was lost by incubation at 45°C for 30min but was stabilized by the addition of DNA. The enzyme contained exonuclease activity but no endonuclease activity. The enzyme produced only light density DNA of various sizes. The function of this enzyme as considered to fill single stranded region of the double stranded primer DNA.     

Purification and properties of 1-phosphofructokinase from Escherichia coli

Abstract The thermophilic facultatively phototrophic green bacterium Chloroflexus aurantiacus strain Ok-70-fl was shown to possess sulfide-repressed hydrogenase activity. Biosynthesis of the enzyme was severely repressed by S²⁻ (5.7 mM) and stimulated specifically by Ni²⁺ and by molecular hydrogen. The hydrogenase was shown to be localized in the cytoplasmic membrane and could be solubilized from the latter by the detergent Triton X-100 in a state forming one enzymatically active band ( M _r 170 × 10³) in polyacrylamide gels. In the membraneous state, the hydrogenase had its maximal activity at 73°C and was active with methyl viologen, methylene blue, menadione and flavins, but not with NAD or NADP as electron acceptors. Solubilization of the enzyme with Triton X-100 resulted in a drastic increase in the FAD/FMN-linked activity.     

Conformational Selection and Substrate Binding Regulate the Monomer/Dimer Equilibrium of the C-terminal domain of Escherichia coli Enzyme I

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by the binding of phosphoenolpyruvate (PEP) to the C-terminal domain (EIC) of enzyme I (EI), a highly conserved protein that is common to all sugar branches of the PTS. EIC exists in a dynamic monomer/dimer equilibrium that is modulated by ligand binding and is thought to regulate the overall PTS. Isolation of EIC has proven challenging, and conformational dynamics within the EIC domain during the catalytic cycle are still largely unknown. Here, we present a robust protocol for expression and purification of recombinant EIC from Escherichia coli and show that isolated EIC is capable of hydrolyzing PEP. NMR analysis and residual dipolar coupling measurements indicate that the isolated EIC domain in solution adopts a stable tertiary fold and quaternary structure that is consistent with previously reported crystallographic data. NMR relaxation dispersion measurements indicate that residues around the PEP binding site and in the β3α3 turn (residues 333-366), which is located at the dimer interface, undergo a rapid transition on the sub-millisecond time scale (with an exchange rate constant of ～1500 s(-1)) between major open (～97%) and minor closed (～3%) conformations. Upon PEP binding, the β3α3 turn is effectively locked in the closed state by the formation of salt bridges between the phosphate group of PEP and the side chains of Lys(340) and Arg(358), thereby stabilizing the dimer.     

Charge Composition Features of Model Single-span Membrane Proteins That Determine Selection of YidC and SecYEG Translocase Pathways in Escherichia coli

We have investigated the features of single-span model membrane proteins based upon leader peptidase that determines whether the proteins insert by a YidC/Sec-independent, YidC-only, or YidC/Sec mechanism. We find that a protein with a highly hydrophobic transmembrane segment that inserts into the membrane by a YidC/Sec-independent mechanism becomes YidC-dependent if negatively charged residues are inserted into the translocated periplasmic domain or if the hydrophobicity of the transmembrane segment is reduced by substituting polar residues for nonpolar ones. This suggests that charged residues in the translocated domain and the hydrophobicity within the transmembrane segment are important determinants of the insertion pathway. Strikingly, the addition of a positively charged residue to either the translocated region or the transmembrane region can switch the insertion requirements such that insertion requires both YidC and SecYEG. To test conclusions from the model protein studies, we confirmed that a positively charged residue is a SecYEG determinant for the endogenous proteins ATP synthase subunits a and b and the TatC subunit of the Tat translocase. These findings provide deeper insights into how pathways are selected for the insertion of proteins into the Escherichia coli inner membrane.     

Proteins from the prokaryotic nucleoid. High-resolution 1H NMR spectroscopic study of Escherichia coli DNA-binding proteins NS1 and NS2

The 1H-NMR spectra of the two Escherichia coli basic, low-Mr (approximately equal to 9000) DNA-binding proteins NS1 and NS2 and of their native complex NS were studied at 400 MHz and a number of resonances and resonance peaks were assigned. As in the case of some eukaryotic histones, the presence of a large number of high-field perturbed Phe resonances, several shielded and deshielded methyl resonances and backbone NH protons quite inaccessible to the solvent clearly indicate the existence of extensive tertiary and, even more so, quaternary structures involving hydrophobic interactions. These structures are lost upon heating, but readily reform upon cooling. Spectral differences between NS1, NS2 and NS and the greater thermal stability of NS indicate that molecules of the heterologous subunits (NS1 and NS2) aggregate (dimerize) preferentially in comparison to the self-aggregation of the homologous subunits. Unlike those of the eukaryotic histones, the tertiary and quaternary structures of NS are insensitive to extensive variations of the ionic strength.     

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.     

The Calcium-Dependent and Calcium-Independent Membrane Binding of Synaptotagmin 1: Two Modes of C2B Binding

The Ca²⁺-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca²⁺-independent manner with a lipid partition coefficient of approximately 3.0 × 10² M^− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca²⁺. Although the Ca²⁺-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca²⁺. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca²⁺, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca²⁺-binding loops of C2B insert into the bilayer. This Ca²⁺-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.