Enzymatic Degradation of Ribosomes During Endogenous Respiration of Pseudomonas aeruginosa.

Gronlund, Audrey F. (University of British Columbia, Vancouver, B.C., Canada), and J. J. R. Campbell. Enzymatic degradation of ribosomes during endogenous respiration of Pseudomonas aeruginosa. J. Bacteriol. 90:1-7. 1965.-From sedimentation analyses it was found that the ribosomal content of Pseudomonas aeruginosa decreased during endogenous respiration. A greater degree of degradation of 50S than 30S ribosomes occurred during the 3-hr starvation period. The enzyme responsible for the initiation of ribosome degradation and present in the ribosome fraction was identified as polynucleotide phosphorylase. The enzyme was inactive in intact 70S ribosomes, but was active in low magnesium ion concentrations which allowed the 70S ribosome to dissociate. Polynucleotide phosphorylase was not solubilized after dissociation of the 70S particle, but remained firmly attached to the 50S and 30S ribosomes, the ribonucleic acid of which served as substrate.     

Variation of the adhesion to polystyrene of phenotypic mutants of Pseudomonas aeruginosa ATCC 27853 during starvation conditions

The aim of this work was to analyse the effects of different growth conditions (phosphate and contemporary carbon-phosphate starvation) on polystyrene adhesion of a strain of Pseudomonas aeruginosa ATCC 27853 and its four phenotypic mutants during experimental growth in starvation conditions. Bacterial adhesion was measured at 20, 40, 60 and 720 min. Data obtained showed that growth conditions are an important factor for the capacity of initial adhesion to inanimate surfaces. The analyses of adhesion of two phenotypic mutants (Mut-P-01 and Mut-P-02) isolated during growth on phosphate starvation is interesting. This kind of experiment yields important information on the prevention of nosocomial infections.     

Phosphate taxis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa was shown to be attracted to phosphate. The chemotactic response was induced by phosphate starvation. The specificity of chemoreceptors for phosphate was high so that no other tested phosphorus compounds elicited a chemotactic response as strong as that elicited by phosphate. Competition experiments showed that the chemoreceptors for phosphate appeared to be different from those for the common amino acids. Mutants constitutive for alkaline phosphatase showed the chemotactic response to phosphate regardless of whether the cells were starved for phosphate.     

Expression in Caulobacter crescentus of the phosphate-starvation-inducible porin OprP of Pseudomonas aeruginosa

The gene for the phosphate-starvation-inducible outer membrane protein OprP, of Pseudomonas aeruginosa was introduced into Caulobacter crescentus CB2A on a plasmid vector. As is the case in P. aeruginosa and Escherichia coli the oprP gene was inducible under conditions of limiting phosphate in C. crescentus. However, the maximal medium concentration of phosphate which still permitted induction of OprP was lower in C. crescentus (50 microM) than in P. aeruginosa (200 microM). Induction of OprP was coincident with the process of stalk elongation, known to occur in C. crescentus under phosphate starvation conditions. When induced, OprP was localized to the cell envelope and became a major membrane protein, indicating that the Pseudomonas promoter was efficiently recognized in C. crescentus and that the gene product was targeted to the appropriate region of the cell. Our data provide support for the hypothesis that the mechanism for regulation of phosphate-starvation-inducible genes is highly conserved amongst the eubacteria.     

Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water.

Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent. In the present study, the survival of P. aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4). Initial counts, before starvation, were the same in all media tested. Following this period, P. aeruginosa became sensitive to PAB with added cetrimide. The addition of FeSO(4) did not improve the recovery of stressed P. aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C. The modified Pseudomonas agar medium was also tested with several P. aeruginosa strains, other species of Pseudomonas, and other genera. Only P. aeruginosa strains (pyocyanin positive) produced the typical colonies. Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P. aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P. aeruginosa in mineral water.     

Choline and betaine as inducer agents of Pseudomonas aeruginosa phospholipase C activity in high phosphate medium

In Pseudomonas aeruginosa, choline or betaine employed as the sole carbon and nitrogen source in a high phosphate medium induced a phospholipase C and an acid phosphatase activity but not an alkaline phosphatase activity. The P. aeruginosa strain utilized in this work does not possess a constitutive phospholipase C, since under culture conditions identical to those utilized by other authors (J. Bacteriol. 93, 670-674 (1967) and J. Bacteriol. 150, 730-738 (1982), our phospholipase C proved to be an inorganic phosphate-repressible enzyme. These findings enable us to conclude that although the phosphate control for the synthesis of phospholipase C may exist, it is expressed only under certain favorable culture conditions.     

Iodine sensitivity of bacteria isolated from iodinated water systems

Fourteen bacterial isolates, predominantly Pseudomonas sp., from two water systems disinfected by iodinated anion-exchange resins were studied and compared with an isolate of Pseudomonas aeruginosa from a povidone-iodine solution and four other isolates. Pseudomonas cepacia and P. aeruginosa grown in brain heart infusion were 3 to 5 logs less sensitive to 1 mg/L I2 (pH 7.2, 1 min) when compared with cultures grown in phosphate buffer. Another P. cepacia isolate was the least sensitive culture when grown in brain heart infusion (1 log decrease) but was more sensitive after cultivation in phosphate buffer (5 logs). Isolates from an iodinated potable water system, including P. cepacia, Staphyloccus warneri, and a Bacillus sp., were all less sensitive to iodine than a "resistant" P. aeruginosa and three other isolates when grown in brain heart infusion. A clinical isolate of P. aeruginosa exhibited intermediate sensitivity. The sensitivity of bacteria to iodine is thus highly variable, depending on the organism as well as the growth conditions.     

Uranyl precipitation by Pseudomonas aeruginosa via controlled polyphosphate metabolism

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.     

Outer membrane porin proteins F, P, and D1 of Pseudomonas aeruginosa and PhoE of Escherichia coli: chemical cross-linking to reveal native oligomers.

Native oligomers of three Pseudomonas aeruginosa outer membrane porin proteins and one Escherichia coli porin were demonstrated by using a chemical cross-linking technique. P. aeruginosa protein F, the major constitutive outer membrane porin, was cross-linked to dimers in outer membrane and whole-cell cross-linking experiments. Purified preparations of P. aeruginosa proteins F, D1 (glucose induced), and P (phosphate starvation induced) and E. coli protein PhoE (Ic) were also cross-linked to reveal dimers and trimers upon two-dimensional sodium dodecyl sulfate-polyacrylamide electrophoretic analysis. Cross-linking of protein F was abolished by pretreatment of the protein with sodium dodecyl sulfate, indicating that the cross-linked products were due to native associations in the outer membrane.     

Phosphoribosylpyrophosphate synthesis from glucose decreases during amino acid starvation of human lymphoblasts

When cultured human lymphoblasts are starved 3 h for an essential amino acid, rates of purine nucleotide synthesis decrease markedly because of a decrease in the intracellular phosphoribosylpyrophosphate concentration (Boss, G.R., and Erbe, R.W. (1982) J. Biol. Chem. 257, 4242-4247; Boss, G. R. (1984) J. Biol. Chem. 259, 2936-2941). In amino acid-starved cells, glucose transport was not changed, whereas total glucose consumption and lactate production decreased by approximately 25 and 10%, respectively. Carbon flow through the oxidative pentose phosphate pathway, measured by 14CO2 release from [1-14C]glucose, decreased by 18% during amino acid starvation. However, kinetic studies of ribulose-5-phosphate 3-epimerase and phosphoriboisomerase suggested that the ribulose 5-phosphate produced by this pathway is converted mostly to xylulose 5-phosphate instead of to ribose 5-phosphate so that this pathway produces little phosphoribosylpyrophosphate. The activity of the nonoxidative pentose phosphate pathway, measured by high performance liquid chromatography following the incorporation of [1-14C]glucose into phosphoribosylpyrophosphate, ATP, and GTP, decreased by approximately 55% during amino acid starvation. None of the enzymes of either pathway changed in specific activity during amino acid starvation. We conclude that the nonoxidative pentose phosphate pathway is the major source of phosphoribosylpyrophosphate for purine nucleotide synthesis and that this pathway is regulated by a metabolite which changes in concentration during amino acid starvation.     

Influence of Carbon or Nitrogen Starvation on Amino Acid Transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa was shown to utilize the majority of commonly occurring amino acids for growth as either the sole carbon or the sole nitrogen source. During carbon or nitrogen deprivation, the rates of transport of most of the amino acids remained unchanged; however, the transport rates for glutamate, alanine, and glycine increased under these conditions and the transport rates for leucine and valine decreased. Normal transport rates for these amino acids were resumed immediately upon the addition of the required nutrient. In the absence of an external source of carbon or of nitrogen, pool amino acids underwent rapid degradation. (14)C-Amino acid pulse experiments indicated that the constitutive amino acid catabolic enzymes, normally present in the organism during growth with glucose as the carbon source, were responsible for rapid pool losses. Nutrient starvation in the presence of chloramphenicol did not prevent amino acid catabolism. This enzymic activity is interpreted as providing P. aeruginosa with a selective advantage for survival during conditions of carbon or nitrogen starvation.     

Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which "natural" conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.     

Influence of starvation for methionine and other amino acids on subsequent bacterial deoxyribonucleic acid replication

Billen, Daniel (University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Tex.), and Roger Hewitt. Influence of starvation for methionine and other amino acids on subsequent bacterial deoxyribonucleic acid replication. J. Bacteriol. 92:609-617. 1966.-A study has been made of the subsequent replicative fate of deoxyribonucleic acid (DNA) synthesized during amino acid starvation by several multiauxotrophic strains of Escherichia coli. Using radioisotopic and density labels and a procedure whereby total cellular DNA is analyzed, we have confirmed and extended a recent report that the DNA made during amino acid starvation behaves anomalously during subsequent DNA replication. When 5-bromouracil (BU) serves as the density lable, 40% or more of the DNA synthesized during starvation will subsequently fail to replicate during three cell generations. Selective amino acid effects were noted. In two methionine-requiring bacteria, methionine deprivation appeared to be of singular importance in influencing the subsequent replicative fate of the DNA made in its absence.When a non-BU density label (N(15), C(13)) was utilized, the effects of amino acid starvation were less obvious. Although the DNA synthesized during complete amino acid starvation in a methionine-requiring E. coli was subsequently more slowly replicated, most of the DNA was finally duplicated during three generations of growth. If methionine was present during starvation for other required amino acids, the subsequent replication rate of the DNA synthesized during this time was more nearly normal, and complete replication was observed. The results have been interpreted as indicating that DNA synthesized during amino acid starvation, and especially during methionine starvation, is somehow altered, and that BU substitution for thymine may interfere with the restoration of such DNA to its replicative state.     

Isolation of atypical lipopolysaccharides from purified cell walls of Pseudomonas cepacia.

Wall fragments were prepared from two strains of Pseudomonas cepacia and from P. aeruginosa, and their contents of readily extractable lipid, pronase-digestible protein and lipopolysaccharide were measured. Lipopolysaccharide extracted from P. cepacia, although biologically active, contained no detectable 2-keto-3-deoxyoctonic acid, but contained phosphate, rhamnose, glucose, heptose and hexosamine in concentrations comparable to those found in P. aeruginosa.     

磷酸饥饿时番茄幼苗酸性磷酸酶活性的变化与Pi吸收的关系

磷酸饥饿时,番茄幼苗根部及地上部酸性磷酸酶活性均显著增强,根部细胞表面酸性磷酸酶及根部外泌的酸性磷酸酶活性亦明显提高。动力学分析表明,磷酸饥饿提高了番茄幼苗根部的酸性磷酸酶对其底物的亲和力。另外,磷酸饥饿对番茄幼苗根部酸性磷酸酶活性的最适ｐＨ值没有影响。钼酸对番茄幼苗根部酸性磷酸酶活性有强烈的抑制作用,对番茄幼苗Ｐｉ吸收速率也有十分明显的抑制效果。以上结果表明,磷酸饥饿时,番茄幼苗Ｐｉ吸收的适应性变化可能与根部酸性磷酸酶特别是根部细胞表面酸性磷酸酶及其外泌酸性磷酸酶的参与密切关联。     

Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp

Phosphate starvation induced oligomeric proteins from the outer membranes of Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aureofaciens, and Pseudomonas chlororaphis were purified to homogeneity. The incorporation of the purified proteins into planar lipid bilayer membranes resulted in stepwise increases in membrane conductance. Single channel conductance experiments demonstrated that these proteins were all capable of forming small channels, similar to the Pseudomonas aeruginosa phospsate porin protein P, with average single channel conductances in 1 M KCl of between 233 and 252 pS. Single channel conductance measurements made in salts of varying cation or anion size indicated that the channels were uniformly anion selective. The measurement of single channel conductance as a function of KCl concentration revealed that all channels saturated at higher salt concentrations, consistent with the presence of an anion-binding site in the channel. Apparent Kd values for Cl- binding were calculated and shown to vary only twofold (180-297 mM) among all channels, including protein P channels. Phosphate competitively inhibited chloride conductance through these channels with apparent I50 values of between 0.59 and 2.5 mM phosphate at 40 mM Cl- and between 9.7 and 27 mM phosphate at 1 m Cl-. These data were consistent with the presence of a phosphate-binding site in the channels of these phosphate-regulated proteins. Furthermore, they indicated that these channels exhibit at least a 20- to 80-fold higher affinity for phosphate than for chloride.     

Quantification of biofilm accumulation by an optical approach

Methods for non-invasive, in situ, measurements of biofilm optical density and biofilm optical thickness were evaluated based on Pseudomonas aeruginosa experiments. Biofilm optical density, measured as intensity reduction of a light beam transmitted through the biofilm, correlates with biofilm mass, measured as total carbon and as cell mass. The method is more sensitive and less labor intensive than other commonly used methods for determining extent of biofilm mass accumulation. Biofilm optical thickness, measured by light microscopy, is translated into physical thickness based on biofilm refraction measurements. Biofilm refractive index was found to be close to the refractive index of water. The P. aeruginosa biofilms studied reached a pseudo steady state in less than a week, with stable liquid phase substrate, cell and TOC concentrations and average biofilm thickness. True steady state was, however, not reached as both biofilm density and roughness were still increasing after 3 weeks.     

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.     

Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal

Abstract We have isolated two alkaline phosphatases (H-AP and L-AP, for high and low molecular mass, respectively) from Pseudomonas aeruginosa PA01. These two enzymes were found to differ in mobility on sodium dodecyl sulphate polyacrylamide gels (H-AP, M _r = 51 000 and L-AP, M _r = 39 500), amino-terminal amino acid sequence and did not cross-react. Both enzymes were active as phosphomonoesterases while only L-AP demonstrated any phosphodiesterase activity. Both enzymes were purified from P. aeruginosa grown in phosphate limiting conditions using the same protocol and were identified in both periplasmic and extracellular locations. A low level of H-AP was produced constitutively whereas L-AP was produced only after induction by reduced phosphate concentration in the growth medium. An L-AP-like enzyme has been previously described, however, this is the first report of a second P. aeruginosa alkaline phosphatase.     

Oxidation of D- and L-valine by enzymes of Pseudomonas aeruginosa

Norton, J. E. (University of Oklahoma School of Medicine, Oklahoma City), and J. R. Sokatch. Oxidation of d- and l-valine by enzymes of Pseudomonas aeruginosa. J. Bacteriol. 92:116-120. 1966.-Cell-free extracts prepared from Pseudomonas aeruginosa grown on dl-valine catalyzed the consumption of oxygen with several d-amino acids, but not with the corresponding l-amino acids. The product of d-valine oxidation was identified as 2-oxoisovalerate by the preparation and characterization of 2-oxoisovalerate 2,4-dinitrophenylhydrazone. The enzyme catalyzing d-amino acid oxidation was present in extracts of cells grown on valine, but not on glucose, had a pH optimum of approximately 9.0, consumed 1 atom of oxygen per mole of keto acid produced, and was not stimulated by any of the usual electron transport cofactors. It was not possible to demonstrate either the direct oxidation of l-valine or the conversion of l- to d-valine by these enzyme preparations. However, a possible route of l-valine metabolism by transamination with 2-oxoglutarate with regeneration of the amino group acceptor by glutamate oxidation was established by identification of the transaminase and l-glutamate dehydrogenase in these enzyme preparations.