Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.     

锌指蛋白的核酸识别特异性研究进展

锌指蛋白是最大的DNA结合蛋白,它能和DNA进行特异性识别,是研究蛋白—DNA相互作用的理想对象。改变锌指元件上的几个保守的氨基酸位点可设计筛选出序列特异的全新锌指蛋白,计算机在锌指蛋白设计方面的应用,使得全新的锌指蛋白识别特异性明显增强。这在基因治疗等方面,具有广阔的应用前景。     

Studies of the cyclic adenosine monophosphate chemoreceptor ofParamecium

Summary A doublet of proteins (48,000M _r) from theParamecium cell body membrane fits several criteria for the external cAMP chemoreceptor. These criteria include: (i) selective elution from a cAMP affinity column, matching a specificity that could be predicted from the behavioral response and whole-cell binding; (ii) binding to wheat germ agglutinin indicating the presence of carbohydrate moieties indicating surface exposure; and (iii) selective inhibition of the intact cells' chemoresponse to cAMP by antibodies against the doublet. Additional evidence for the existence of a receptor, in general, comes from selective elimination of the cAMP chemoresponse by photoaffinity labeling of whole cells with 8-N₃-cAMP. The doublet proteins are not identical to the regulatory subunit of a cAMP-dependent protein kinase fromParamecium, theDictyostelium cAMP chemoreceptor, or the 42–45 kDa range proteins related to the large surface glycoprotein inParamecium. The doublet proteins are not readily separable and, as inDictyostelium, may represent two different covalent modification states of the same protein. Amino acid analysis indicates that the proteins are similar, but does not distinguish between the possibilities of proteolysis and covalent modification. Once cloned, this doublet may prove to be only the fifth external, eukaryotic chemoreceptor to be identified.     

Transmembrane organization of the Bacillus subtilis chemoreceptor McpB deduced by cysteine disulfide crosslinking

The Bacillus subtilis chemoreceptor McpB is a dimer of identical subunits containing two transmembrane (TM) segments (TM1, residues 17-34: TM2, residues 280-302) in each monomer with a 2-fold axis of symmetry. To study the organization of the TM domains, the wild-type receptor was mutated systematically at the membrane bilayer/extracytoplasmic interface with 15 single cysteine (Cys) substitutions in each of the two TM domains. Each single Cys substitution was capable of complementing a null allele in vivo, suggesting that no significant perturbation of the native tertiary or quaternary structure of the chemoreceptor was introduced by the mutations. On the basis of patterns of disulfide crosslinking between subunits of the dimeric receptor, an alpha-helical interface was identified between TM1 and TM1' (containing residues 32, 36, 39, and 43) and between TM2 and TM2' (containing residues 276, 277, 280, 283 and 286). Pairs of cysteine substitutions (positions 34/280 and 38/273) in TM1 and TM2 were used to further elucidate specific contacts within a monomer subunit, enabling a model to be constructed defining the organization of the TM domain. Crosslinking of residues that were 150-180 degrees removed from position 32 (positions 37, 41, and 44) suggested that the receptors may be organized as an array of trimers of dimers in vivo. All crosslinking was unaffected by deletion of cheB and cheR (loss of receptor demethylation/methylation enzymes) or by deletion of cheW and cheV (loss of proteins that couple receptors with the autophosphorylating kinase). These findings indicate that the organization of the transmembrane region and the stability of the quaternary complex of receptors are independent of covalent modifications of the cytoplasmic domain and conformations in the cytoplasmic domain induced by the coupling proteins.     

Modulation of duck intrapulmonary chemoreceptor discharge by cardiac activity

Summary A previous study on duck intrapulmonary chemoreceptors (IPC) reported that the cardiac cycle modulated discharge of most IPC, but did not distinguish whether pulsatile blood flow or cardiac-linked fluctuation in parabronchial gas flow was responsible. In this study with Pekin ducks one of 14 IPC recorded was found to be modulated by cardiac activity in the perfused lung, and none in the non-perfused lung. Discharge variability expressed as coefficients of variation did not change with pulmonary artery ligation. It is concluded that: (1) pulsatile blood flow does not affect discharge regularity of most IPC and (2) the infrequent occurrence of cardiac modulation of IPC in thoracotomized ducks may be the result of IPC having receptive endings near the parabronchial lumen.     

The Aer2 chemoreceptor from Vibrio vulnificus is a tri-PAS-heme oxygen sensor

The marine pathogen Vibrio vulnificus senses and responds to environmental stimuli via two chemosensory systems and 42–53 chemoreceptors. Here, we present an analysis of the V. vulnificus Aer2 chemoreceptor, VvAer2, which is the first V. vulnificus chemoreceptor to be characterized. VvAer2 is related to the Aer2 receptors of other gammaproteobacteria, but uncharacteristically contains three PAS domains (PAS1-3), rather than one or two. Using an E. coli chemotaxis hijack assay, we determined that VvAer2, like other Aer2 receptors, senses and responds to O₂. All three VvAer2 PAS domains bound pentacoordinate b-type heme and exhibited similar O₂ affinities. PAS2 and PAS3 both stabilized O₂ via conserved Iβ-Trp residues, but PAS1, which was easily oxidized in vitro, was unaffected by Iβ-Trp replacement. Our results support a model in which PAS1 is largely dispensable for O₂-mediated signaling, whereas PAS2 modulates PAS3 signaling, and PAS3 signals to the downstream domains. Each PAS domain appeared to be positionally optimized, because PAS swapping caused altered signaling properties, and neither PAS1 nor PAS2 could replace PAS3. Our findings strengthen previous conclusions that Aer2 receptors are O₂ sensors, but with distinct N-terminal domain arrangements that facilitate, modulate and tune responses based on environmental signals.     

Physiologically relevant divalent cations modulate citrate recognition by the McpS chemoreceptor

The McpS chemoreceptor of Pseudomonas putida KT2440 recognizes six different tricarboxylic acid (TCA) cycle intermediates. However, the magnitude of the chemotactic response towards these compounds differs largely, which has led to distinguish between strong attractants (malate, succinate, fumarate, oxaloacetate) and weak attractants (citrate, isocitrate). Citrate is abundantly present in plant tissues and root exudates and can serve as the only carbon source for growth. Citrate is known to form complexes with divalent cations which are also abundantly present in natural habitats of this bacterium. We have used isothermal titration calorimetry to study the formation of citrate-metal ion complexes. In all cases binding was entropy driven but significant differences in affinity were observed ranging from K(D)=157 μM (for Mg(2+)) to 3 μM (for Ni(2+)). Complex formation occurred over a range of pH and ionic strength. The ligand binding domain of McpS (McpS-LBD) was found to bind free citrate, but not complexes with physiologically relevant Mg(2+) and Ca(2+). In contrast, complexes with divalent cations which are present as trace elements (Co(2+), Cd(2+) and Ni(2+)) were recognized by McpS-LBD. This discrimination differs from other citrate sensing proteins. These results are discussed in the context of the three dimensional structure of free citrate and its complex with Mg(2+). Chemotaxis assays using P. putida revealed that taxis towards the strong attractant malate is strongly reduced in the presence of free citrate. However, this reduction is much less important in the presence of citrate-Mg(2+) complexes. The physiological relevance of these findings is discussed.     

Site-directed spin labeling of a bacterial chemoreceptor reveals a dynamic, loosely packed transmembrane domain

We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to investigate dynamics and helical packing in the four-helix transmembrane domain of the homodimeric bacterial chemoreceptor Trg. We focused on the first transmembrane helix, TM1, particularly on the nine-residue sequence nearest the periplasm, because patterns of disulfide formation between introduced cysteines had identified that segment as the region of closest approach among neighboring transmembrane helices. Along this sequence, mobility and accessibility of the introduced spin label were characteristic of loosely packed or solvent-exposed side chains. This was also the case for eight additional positions around the circumference and along the length of TM1. For the continuous nine-residue sequence near the periplasm, mobility and accessibility varied only modestly as a function of position. We conclude that side chains of TM1 that face the interior of the four-helix domain interact with neighboring helices but dynamic movement results in loose packing. Compared to transmembrane segments of other membrane proteins reconstituted into lipid bilayers and characterized by site-directed spin labeling, TM1 of chemoreceptor Trg is the most dynamic and loosely packed. A dynamic, loosely packed chemoreceptor domain can account for many experimental observations about the transmembrane domains of chemoreceptors.     

Relative air humidity influences the function of the tarsal chemoreceptor cells of the cherry fruit fly (Rhagoletis cerasi)

ABSTRACT. Electrophysiological recordings from the tarsal D-sensilla of mature, 5–7-day-old female European cherry fruit fly ( Rhagoletis cerasi L.) revealed that a large proportion of sensilla showed no sensory activity in response to stimulation with the oviposition deterring pheromone ('ODP') or sucrose. In contrast, good recordings were obtained from almost all sensilla in freshly emerged, 1-day-old flies. Ageing, nutritional state and contamination were excluded as possible major factors influencing the responsiveness of the D-sensilla. The study of the variability in responsiveness of the sensilla of 1-day-old flies among different tarsomers, among tarsi of the same individual and among individuals, revealed that the major source of variability was among individuals.
Individual flies were also affected by the humidity in the holding cages: relative air humidity of less then 75% for 4–6 days was shown to reduce the number of D-sensilla giving recordings with a good signal-to-noise ratio. The D-sensilla on the most distal tarsomers were affected most by the reduced (30–55% r. h.) air humidity. Flies which had contact with plant leaves showed less reduction in responsiveness at low air humidity (40% r. h.) then flies without leaf contact.     

Crystal structures of bacterial FabH suggest a molecular basis for the substrate specificity of the enzyme

FabH (β-ketoacyl-acyl carrier protein synthase III) is unique in that it initiates fatty acid biosynthesis, is inhibited by long-chain fatty acids providing means for feedback control of the process, and dictates the fatty acid profile of the organism by virtue of its substrate specificity. We report the crystal structures of bacterial FabH enzymes from four different pathogenic species: Enterococcus faecalis, Haemophilus influenzae, Staphylococcus aureus and Escherichia coli. Structural data on the enzyme from different species show important differences in the architecture of the substrate-binding sites that parallel the inter-species diversity in the substrate specificities of these enzymes.     

Molecular mechanism of substrate specificity in the bacterial neutral amino acid transporter LeuT

The recently published X-ray structure of LeuT, a Na(+)/Cl(-)-dependent neurotransmitter transporter, has provided fresh impetus to efforts directed at understanding the molecular principles governing specific neurotransmitter transport. The combination of the LeuT crystal structure with the results of molecular simulations enables the functional data on specific binding and transport to be related to molecular structure. All-atom FEP and molecular dynamics (MD) simulations of LeuT embedded in an explicit membrane were performed alongside a decomposition analysis to dissect the molecular determinants of the substrate specificity of LeuT. It was found that the ligand must be in a zwitterionic (ZW) form to bind tightly to the transporter. The theoretical results on the absolute binding-free energies for leucine, alanine, and glycine show that alanine can be a potent substrate for LeuT, although leucine is preferred, which is consistent with the recent experimental data (Singh et al., Nature 2007;448:952-956). Furthermore, LeuT displays robust specificity for leucine over glycine. Interestingly, the ability of LeuT to discriminate between substrates relies on the dynamics of residues that form its binding pocket (e.g., F253 and Q250) and the charged side chains (R30-D404) from a second coordination shell. The water-mediated R30-D404 salt bridge is thought to be part of the extracellular (EC) gate of LeuT. The introduction of a polar ligand such as glycine to the water-depleted binding pocket of LeuT gives rise to structural rearrangements of the R30-D404-Q250 hydrogen-bonding network and leads to increased hydration of the binding pocket. Conformational changes associated with the broken hydrogen bond between Q250 and R30 are shown to be important for tight and selective ligand binding to LeuT.     

Tuning a bacterial chemoreceptor with protein-membrane interactions

Chemoreceptors in Escherichia coli are homodimeric transmembrane proteins that convert environmental stimuli into intracellular signals controlling flagellar motion. Chemoeffectors bind to the extracellular (periplasmic) domain of the receptors, whereas their cytoplasmic domain mediates signaling and adaptation. The second transmembrane helix (TM2) connects these two domains. TM2 contains an aliphatic core flanked by amphipathic aromatic residues that have specific affinity for polar-hydrophobic membrane interfaces. We previously showed that Trp-209, near the cytoplasmic end of TM2, helps maintain the normal baseline-signaling state of the aspartate chemoreceptor (Tar) and that Tyr-210 plays an auxiliary role in this control. We have now repositioned the Trp-209/Tyr-210 pair in single-residue increments about the cytoplasmic polar-hydrophobic interface. Changes from WY-2 to WY+1 modulate the baseline-signaling state of the receptor in predictable and incremental steps that can be compensated by adaptive methylation/demethylation. Greater displacements, as in WY-3, WY+2, and WY+3, bias the receptor to the off kinase-inhibiting state or the on kinase-stimulating state, respectively, to a degree that cannot be fully compensated by the adaptation system. Aromatic residues analogous to Trp-209/Tyr-210 are present in other chemoreceptors and many transmembrane sensor kinases, where they may serve a similar function.     

Altered product specificity of a cyclodextrin glycosyltransferase by molecular imprinting with cyclomaltododecaose

Cyclodextrin glycosyltransferases (CGTases), members of glycoside hydrolase family 13, catalyze the conversion of amylose to cyclodextrins (CDs), circular α‐(1,4)‐linked glucopyranose oligosaccharides of different ring sizes. The CD containing 12 α‐D‐glucopyranose residues was preferentially synthesized by molecular imprinting of CGTase from Paenibacillus sp. A11 with cyclomaltododecaose (CD₁₂) as the template molecule. The imprinted CGTase was stabilized by cross‐linking of the derivatized protein. A high proportion of CD₁₂ and larger CDs was obtained with the imprinted enzyme in an aqueous medium. The molecular imprinted CGTase showed an increased catalytic efficiency of the CD₁₂‐forming cyclization reaction, while decreased k_cat/K_m values of the reverse ring‐opening reaction were observed. The maximum yield of CD₁₂ was obtained when the imprinted CGTase was reacted with amylose at 40°C for 30 min. Molecular imprinting proved to be an effective means toward increase in the yield of large‐ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations. Copyright © 2010 John Wiley & Sons, Ltd.     

田菁根瘤菌YIC4027可溶性趋化受体Tlp1的功能鉴定

【目的】初步探究田菁根瘤菌Sinorhizobium alkalisoli YIC4027中唯一含有PAS结构域可溶性趋化受体Tlp1的功能机理。【方法】本研究基于Red重组系统以及三亲接合技术进行缺失突变株的构建。对野生型和突变株的生长情况、趋化能力、趋氧性、细胞凝结、生物膜的形成、胞外多糖产量、在宿主根表的定殖及竞争性结瘤等表型进行了测定。【结果】与野生型相比,突变株的生长不受影响,趋化和趋氧能力降低,在宿主根表的定殖及竞争性结瘤能力降低,而细胞凝结能力、生物膜形成以及胞外多糖产生能力等均有所提高【。结论】本研究首次证实了S. alkalisoli YIC4027中可溶性趋化受体Tlp1影响细胞的趋化运动。     

封闭循环养殖系统中β变形菌亚纲氨氧化细菌的引物特异性

摘要:【目的】为探讨4 对不同的引物对封闭循环养殖系统生物膜中β 变形菌亚纲氨氧化细菌(β-AOB) 的特异性差异。【方法】采用16S rDNA 文库克隆技术对β-AOB 的多样性进行了分析。【结果】以引物CTO189f/CTO654r 扩增构建的文库中所含β-AOB 比例最高,可达67. 3%。不同封闭循环养殖系统的生物膜对引物的扩增效率有明显的影响,其中以养殖尼罗罗非鱼的封闭循环养殖系统生物膜为DNA 来源的,引物均有较高的扩增效率。【结论】针对封闭循环养殖系统生物膜中的β-AOB,特异性最高的是CTO189f /CTO654r 引物。     

RFLP mapping and race specificity of bacterial blight resistance genes (QTLs) in rice

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Studies on the specificity of acetylaminoacyl-peptide hydrolase.

In a continuing attempt to explore the types of specificity determinants that may affect protein-protein (peptide) interactions, a number of short (2-5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl-peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac-AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat for the different substrates were determined by standard steady-state kinetics, and the corresponding delta delta GT++ value derived from kcat/Km was used for the comparison, setting delta detal GT++ for Ac-AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1', 2', or 3' (delta delta GT++ values of 2-5 kJ); the negative charge provided by the C-terminus of the substrate also appears to be important, since the amide and O-Me ester derivatives caused a change in delta delta GT++ values of -7 to -8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582-589), and the proposed active site model incorporates subsites for both charge-charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make-up of each peptide and not only the effect of a single residue in a given position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the substrate specificity of the bacterial Pnkp/Hen1 RNA repair system using synthetic RNAs

Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed.     

Substrate specificity of the erythrocyte Ca2+-ATPase

In the absence of Mg²⁺, the observed activity of the erythrocyte plasma membrane Ca²⁺-ATPase is due to the hydrolysis of CaATP at a low rate. In the presence of Mg²⁺, the activity of the enzyme is much higher, but it is inhibited by high levels of free Mg²⁺. This inhibition appears to be due to competition of Mg²⁺ and Ca²⁺ for a site on the enzyme, rather than for ATP.