Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase

Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Expression of the phospholipase C gene of Pseudomonas aeruginosa in Escherichia coli and Pseudomonas

The plc gene for phospholipase of Pseudomonas aeruginosa, able to be transcribed only from its own promoter, has been introduced into Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida cells in the recombinant plasmid pPMS21 of a wide host range. The expression of plc gene in all recipient cells has been shown to be phosphate regulated. The fact emphasizes the identity of pho-regulation systems in Escherichia coli and Pseudomonas cells. The level of phospholipase activity is similar in Pseudomonas putida and Pseudomonas aeruginosa under the conditions of the gene derepression, while in Escherichia coli cells the level does not exceed 10% of activity registered in Pseudomonas cells.     

Overexpression of a thermostable lipase gene from Pseudomonas fluorescens in Escherichia coli

Summary A thermostable lipase gene from Pseudomonas fluorescens SIK W1 was overexpressed in Escherichia coli BL21 using expression vector pTTY2. The amount of lipase produced by E. coli BL21 with pTTY2 was more than 40% of the total cell proteins when induced with isopropyl--d-thiogalactopyranoside. The lipase was produced as inclusion bodies in the cytoplasm of E. coli. They were solubilized by 8 m urea and refolded into biologically active form. The refolded lipase showed high thermostability; the time required for 90% inactivation of the enzyme (D-value) was 4 h at 95°C and the increment of temperature to reduce heating times by 90% (z _d value) was 76°C.Offprint requests to: J. S. Rhee     

Pseudomonas aeruginosa amidase: aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonas aeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of soluble amidase, relatively to other proteins, was obtained in the condition at 37°C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all the tested growth conditions. Addition of ethanol at 25°C in the culture medium improved amidase yield, which quantitatively aggregated in a biological active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37°C by FTIR analysis demonstrated a lower content of intermolecular interactions which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.     

Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.     

Overexpression in Escherichia coli and functional analysis of a novel PPi-selective porin, oprO, from Pseudomonas aeruginosa.

Immediately upstream from and adjacent to the oprP gene, which codes for the phosphate-specific porin OprP of Pseudomonas aeruginosa, lies the PR region (oprO), which cross-hybridizes with oprP DNA. To determine the function of this region, the oprO gene was expressed behind the lactose promoter in Escherichia coli, and the resultant OprO protein was purified and reconstituted into planar lipid bilayers. OprO formed sodium dodecyl sulfate-stable trimers, cross-reacted immunologically with OprP, and, like OprP, formed an anion-specific, phosphate-selective porin. However, it demonstrated lower affinity for and higher maximal conductance of both chloride and phosphate than did the OprP channel. Examination by macroscopic conductance inhibition experiments of the affinity of OprO for phosphates of different lengths revealed a preference for PPi and tripolyphosphate over Pi, suggesting that OprO functioned as a PPi-selective polyphosphate channel, in contrast to OprP, which has a marked preference for Pi.     

Cloning and expression of Pseudomonas aeruginosa flagellin in Escherichia coli.

The flagellin gene was isolated from a Pseudomonas aeruginosa PAO1 genomic bank by conjugation into a PA103 Fla- strain. Flagellin DNA was transferred from motile recipient PA103 Fla+ cells by transformation into Escherichia coli. We show that transformed E. coli expresses flagellin protein. Export of flagellin to the E. coli cell surface was suggested by positive colony blots of unlysed cells and by isolation of flagellin protein from E. coli supernatants.     

利用抗生素对大肠埃希菌和铜绿假单胞菌的影响进行菌株区分

目的为了探讨抗生素对中心碳代谢的影响,我们研究了大肠埃希菌和铜绿假单胞菌在11种不同抗生素的刺激下,三羧酸循环相关有机酸的代谢变化。方法利用毛细管电泳技术对2种菌在不同抗生素作用下细胞内的主要有机酸进行检测,然后通过多变量统计分析对数据进行处理。结果通过多变量统计分析发现,2种细菌可以通过抗生素对其胞内有机酸的影响不同而得到区分。结论胞内有机酸的变化具有菌株特异性,可以用于细菌的区分。     

Sensitivity of Escherichia coli and Pseudomonas aeruginosa to avoparcin, flavomycin and virginiamycin

Abstract The sensitivities of wild-type, rough, deep-rough and antibiotic-hypersensitive strains of Escherichia coli and Pseudomonas aeruginosa to avoparcin, flavomycin and virginiamycin are described. Avoparcin was least active against these strains. Overall, the results suggested that resistance of wild-type cells to these antibiotics resulted from their exclusion from the cells.     

Elongation Factor P is Dispensable in Escherichia coli and Pseudomonas aeruginosa

3-Hydroxypropionic acid (3-HP) is a commercially important platform chemical from which a panel of chemicals can be generated. Klebsiella pneumoniae has been regarded as a promising host strain in glycerol-based 3-HP production for its exceptional ability to metabolize glycerol. Since the glycerol dissimilation mechanism governs the carbon flux distribution from glycerol, inducible strong promoters were usually employed to enhance the glycerol consumption and 3-HP production. Here, we report an alternative strategy that the native promoter of dhaB gene was applied to enhance 3-HP production in K. pneumoniae. The key enzyme genes (ald4 and dhaB) for 3-HP biosynthesis were co-expressed under this promoter. Metabolic analysis revealed that the 3-HP formation was partially coupled with cell metabolism. To optimize the production of 3-HP, the effects of glucose as energy source assistant were investigated based on the analysis of fermentation process kinetics. The highest 3-HP yield (3.77 g/L in flask) was observed upon optimized conditions. Since there were no additional inducers needed, the strategy of employing native promoter seems more feasible to industrial application. More importantly, the employment of constitutive promoter demonstrated an effective approach for decoupling the natural correlation between respiratory metabolism and glycerol dissimilation in K. pneumoniae.     

Regulation of components of the Pseudomonas aeruginosa phosphate-starvation-inducible regulon in Escherichia coli

Plasmids pPBP and pRS-XP containing the cloned genes for the Pseudomonas aeruginosa phosphate-starvation-inducible periplasmic phosphate-binding protein and outer membrane porin P (oprP), respectively, were introduced into various Escherichia coli Pho-regulon regulatory mutants. Using Western immunoblots and specific antisera, the production of both gene products was observed to be under the control of regulatory elements of the E. coli Pho regulon. Sequencing of the region upstream of the translational start site of the oprP gene revealed a 'Pho box' with strong homology to the E. coli consensus 'Pho box', the putative binding site of the PhoB activator. Since P. aeruginosa and E. coli belong to different families and have quite different GC contents, these data suggest strong evolutionary conservation of regulatory elements of the Pho regulon.     

Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli.

Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.     

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.     

Morphological alterations of Pseudomonas aeruginosa and Escherichia coli by nocardicin A

Abstract Pseudomonas aeruginosa and Escherichia coli were exposed to nocardicin A, and were subsequently observed with transmission and scanning electron microscopes. Although the nocardicin A-induced morphological alterations such as bulges and spheroplast formations were observed both in P. aeruginosa and E. coli , their positions on the cell surface were different in the two species.