RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS

Bacterial signaling histidine kinases (HKs) have long been postulated to function exclusively through linear signal transduction chains. However, several HKs have recently been shown to form complex multikinase networks (MKNs). The most prominent MKN, involving the enzymes RetS and GacS, controls the switch between the motile and biofilm lifestyles in the pathogenic bacterium Pseudomonas aeruginosa. While GacS promotes biofilm formation, RetS counteracts GacS using three distinct mechanisms. Two are dephosphorylating mechanisms. The third, a direct binding between the RetS and GacS HK regions, blocks GacS autophosphorylation. Focusing on the third mechanism, we determined the crystal structure of a cocomplex between the HK region of RetS and the dimerization and histidine phosphotransfer (DHp) domain of GacS. This is the first reported structure of a complex between two distinct bacterial signaling HKs. In the complex, the canonical HK homodimerization interface is replaced by a strikingly similar heterodimeric interface between RetS and GacS. We further demonstrate that GacS autophosphorylates in trans, thus explaining why the formation of a RetS-GacS complex inhibits GacS autophosphorylation. Using mutational analysis in conjunction with bacterial two-hybrid and biofilm assays, we not only corroborate the biological role of the observed RetS-GacS interactions, but also identify a residue critical for the equilibrium between the RetS-GacS complex and the respective RetS and GacS homodimers. Collectively, our findings suggest that RetS and GacS form a domain-swapped hetero-oligomer during the planktonic growth phase of P. aeruginosa before unknown signals cause its dissociation and a relief of GacS inhibition to promote biofilm formation.     

Insights into the atypical autokinase activity of the Pseudomonas aeruginosa GacS histidine kinase and its interaction with RetS

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Adenylate kinase as a virulence factor of Pseudomonas aeruginosa

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.     

The intracellular localization of Pseudomonas aeruginosa lectins

The localization of the Pseudomonas aeruginosa lectins (PA-I and PA-II) was studied using methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release of the two lectins occurred when P. aeruginosa was exposed to magnesium-osmotic shock or was frozen and thawed. Under these conditions, release of the periplasmic 5'-nucleotidase occurred, whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected. Formation of spheroplasts from P. aeruginosa by gradual removal of the bacterial envelopes revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme treated mureinoplasts resulted in low release of glucose-6-phosphate dehydrogenase and the two lectins (10-13%) and a considerable activity (38.4%) of 5'-nucleotidase. The presence of the lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P. aeruginosa to agglutinate papain-treated human erythrocytes. These results indicate that the two lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer membranes and in the periplasmic space.     

Polar localization of a soluble methyl-accepting protein of Pseudomonas aeruginosa

A soluble methyl-accepting chemotaxis protein (MCP) of Pseudomonas aeruginosa, McpS, showed polar localization by immunofluorescence microscopy. Overexpression of McpS resulted in a dominant-negative effect on chemotaxis and caused a loss of polar clustering of the general MCP population. The polar localization of a soluble MCP defines a third, and unexpected, paradigm for cellular MCP localization.     

Localization of cholinesterase in Pseudomonas aeruginosa strain K

The inducible cholinesterase of Pseudomonas aeruginosa strain K (ATCC 25102) degraded propionylcholine, acetylthiocholine, acetylcholine and acetyl-beta-methylcholine at a high rate and butyrylcholine and succinylcholine at very low rates. The localization of the enzyme in the periplasmic space was indicated by a similar rate of acetylcholine degradation by intact cells or their extracts, by release of cholinesterase together with alkaline phosphatase into the culture medium during cell growth in a low phosphate-containing medium, by liberation of cholinesterase and alkaline phosphatase during lysozyme-induced conversion of cells to spheroplasts and by freezing and thawing. Threatment of cells with diazo-7-amino-1,3-naphthalenedisulphonic acid, which inactivates surface-located enzymes, abolished most of the cholinesterase and 5'-nucleotidase activities.     

铜绿假单胞菌噬菌体PaP4的分离与鉴定

[目的]鉴定一株新分离的铜绿假单胞菌噬菌体PaP4的生物学特性.[方法]双层琼脂培养法制备PaP4的单个噬斑,观察噬斑特点;用聚乙二醇8000浓缩PaP4颗粒后,再用氯化铯密度梯度离心纯化;用透射电子显微镜观察磷钨酸负染色的PaP4颗粒;提取PaP4基因组核酸,通过限制性内切酶图谱分析其核酸类型;按照感染复数(MOI)分别为0.000 1、0.001、0.01、0.1、1和10加入噬菌体纯培养液和宿主菌,充分裂解细菌后,测定噬菌体滴度;以MOI=10的比例加入噬菌体及宿主菌,进行一步生长实验,绘制一步生长曲线.[结果]PaP4的噬斑直径约3 mm-5 mm,圆形透明边缘清晰;PaP4噬菌体呈多面体立体对称的头部,直径约50 nm,有一个约30 nm的短尾;限制性酶切实验表明PaP4基因组为双链DNA;当MOI为0.001时PaP4感染其宿主菌产生的子代噬菌体滴度最高;用一步生长曲线描绘了其生长特性.[结论]PaP4属dsDNA短尾科裂解性噬菌体;最佳感染复数是0.001;由一步生长曲线得出感染宿主菌的潜伏期是25 min,裂解期是20 min,平均裂解量是150.     

Properties and localization of N-acetylglutamate deacetylase from Pseudomonas aeruginosa

The N-acetylglutamate deacetylase (EC 3.5.1.-) from Pseudomonas aeruginosa, strain PAO1, was purified 15,000-fold to electrophoretic homogeneity. The enzyme was distinct from acetylornithinase and formylglutamate hydrolase. Its molecular weight was estimated to be 90,000 by gel filtration and by sedimentation in sucrose gradients. Electrophoresis in sodium-dodecyl sulphate gels gave a single band corresponding to a molecular weight of 44,000. N-Acetylglutamate deacetylase was L-specific and showed no peptidase activity. Among 17 N-acetyl-L-amino acids tested as substrates, N-acetyl-L-glutamine, N-acetyl-L-methionine and N-acetylglycine were hydrolysed at 20% of the rate of N-acetyl-L-glutamate whereas other N-acetyl-L-amino acids were deacetylated at a rate of less than 10%. The catalytic activity depended on Co2+. The Km of the enzyme with respect to N-acetylglutamate was 1.43 mM. Preparation of spheroplasts with lysozyme in the presence of 0.2 M-MgCl2 led to the release of 80% of the enzyme activity from the cells, indicating the periplasmic localization of N-acetylglutamate deacetylase. Its localization in the periplasmic space explains the inability of P. aeruginosa argA mutants to grow on N-acetylglutamate, which is utilized by the wild-type as a carbon and nitrogen source.     

Improved PCR for identification of Pseudomonas aeruginosa

The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41?×?10³?copies/μl of cloned amplified target DNA using purified DNA, or 2.7?×?10² colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.     

The subcellular localization of a C-terminal processing protease in Pseudomonas aeruginosa

Carboxy (C)-terminal processing proteases (CTP) are a relatively new group of serine proteases. Found in a broad range of organisms - bacteria, archaea, algae, plants and animals - these proteases are involved in the C-terminal processing of proteins. In comparison with amino-terminal processing of bacterial proteins, less is known about C-terminal processing and its physiological function. Bacterial CTPs appear to influence different basal cellular processes. Although CTPs of Gram-negative bacteria are generally referred to as being localized in the periplasm, there is little experimental evidence for this. We show for the first time the subcellular localization of a CTP-3 family protein from Pseudomonas aeruginosa, named CtpA, in the periplasm by a carefully designed fractionation study. Our results provide experimental evidence for the generally accepted hypothesis that CTPs are located in the periplasmic space of Gram-negative bacteria.