Ornibactins—a new family of siderophores from Pseudomonas

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn¹(N -OH, N -acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn⁴(N -OH, N -formyl)-1,4-diaminobutane. The N -acyl groups of Orn¹(N -OH, N -acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.     

The Pseudomonas aeruginosa membranes: a target for a new amphiphilic aminoglycoside derivative?

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.     

The BapF protein from Pseudomonas aeruginosa is a β-peptidyl aminopeptidase

A gene encoding a so far uncharacterized β-peptidyl aminopeptidase from the opportunistic human pathogen Pseudomonas aeruginosa PAO1 was cloned and actively expressed in the heterologue host Escherichia coli. The gene was identified in the genome sequence by its homology to the S58 family of peptidases. The sequence revealed an open reading frame of 1,101 bp with a deduced amino acid sequence of 366 amino acids. The gene was amplified by PCR, ligated into pET22b(+) and was successfully expressed in E. coli BL21 (DE3). It was shown that the enzyme consists of two polypeptides (α- and β-subunit), which are processed from the precursor. The enzyme is specific for N-terminal β-alanyl dipeptides (β-Ala-Xaa). BapF hydrolyses efficiently β-alanine at the N-terminal position, including H-β³hAla-pNA, H–D-β³hAla-pNA and β-Ala-l-His (l-carnosine). d- and l-alaninamide were also hydrolysed by the enzyme.     

Multi-drug resistant Pseudomonas aeruginosa: towards a therapeutic dead end?

Pseudomonas aeruginosa is a major hospital-associated pathogen that can cause severe infections, most notably in patients with cystic fibrosis or those hospitalized in intensive care units. In this context, the current increase in incidence of multi-drug resistant (MDR) isolates of P. aeruginosa (MDRPA) raises serious concerns. MDR in P. aeruginosa is defined as the resistance to 3 or 4 of the following antibiotic classes: penicillins/cephalosporins/monobactams, carbapenems, aminoglycosides, and fluoroquinolones. These strains constantly cumulate several resistance mechanisms as a consequence of multiple genetic events, i.e., chromosomal mutations or horizontal transfers of resistance genes. Involved mechanisms may include active efflux, impermeability resulting from porins loss, plasmid-encoded b-lactamases/carbapenemases or aminoglycosides-modifying enzymes, and enzymatic or mutation-associated changes in antibiotics targets. Antibiotic selection pressure represents the leading risk factor for MDRPA acquisition. Colistin (polymyxin E) remains active on virtually all MDRPA isolates, and increasingly appears as the last available option to treat infections caused by these strains. However, the emergence of colistin resistance has been reported in P. aeruginosa, which may announce the spread of pan-resistant strains in a close future.     

Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

Structure-function relationship of type-IV prepilin peptidase of Pseudomonas aeruginosa – a review

The bifunctional enzyme prepilin peptidase (PilD) from Pseudomonas aeruginosa is a key determinant in both type-IV pilus biogenesis and extracellular protein secretion, in its roles as a leader peptidase and MTase. It is responsible for endopeptidic cleavage of the unique leader peptides that characterize type-IV pilin precursors, as well as proteins with homologous leader sequences that are essential components of the general secretion pathway found in a variety of Gram-negative pathogens. Following removal of the leader peptides, the same enzyme is responsible for the second posttranslational modification that characterizes the type-IV pilins and their homologues, namely N-methylation of the newly exposed N-terminal amino acid residue. This review discusses some of the work begun in order to answer questions regarding the structure-function relationships of the active sites of this unique enzyme.     

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of Achromobacter cycloclastes nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd ₁ nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d ₁, indicating that both heterologous hosts synthesized a cytochrome cd ₁ without the d ₁-group.Abbreviations Cu-NIR Cu-containing nitrite reductase - DDC diethyldithiocarbamate - EPR electron paramagnetic resonance - IPTG isopropyl--D-galactoside - SDS sodium dodecyl sulfate - LB medium Luria-Bertani medium     

Phenogenetic Characterization of a Group of Giant φKZ-like Bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group ofPseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage KZ genome (288 334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1–19]. By DNA homology, the phages were assigned to three species (represented by phages KZ, Lin68, and EL, respectively) and two new genera (KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the KZ species (KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the KZ genus. The origin and evolution of the KZ-like phages are discussed. Analysis of protein sequences encoded by the phage KZ genome made it possible to assume wide migration of the KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two β-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G_M1 and asialo-G_M2 and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAcβ(1-4)Gal, common in both asialo-G_M1 and asialo-G_M2, likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.     

Purification and biochemical characterization of a detergent stable α-amylase from Pseudomonas stutzeri AS22

This study reports the purification and biochemical characterization of a novel maltotetraose-forming-α-amylase from Pseudomonas stutzeri AS22, designated PSA. The P. stutzeri α-amylase (PSA) was purified from the culture supernatant to homogeneity by Sepharose mono Q anion exchange chromatography, ultrafiltration and Sephadex G-100 gel filtration, with a 37.32-fold increase in specific activity, and 31% recovery. PSA showed a molecular weight of approximately 57 kDa by SDS-PAGE. The N-terminal amino acid sequence of the first 7 amino acids was DQAGKSP. This enzyme exhibited maximum activity at pH 8.0 and 55°C, performed stably over a broad range of pH 5.0 ≈ 12.0, but rapidly lost activity above 50°C. Both potato starch and Ca²⁺ ions have a protective effect on the thermal stability of PSA. The enzyme activity was inhibited by Hg²⁺, Mn²⁺, Cd²⁺, Cu²⁺, and Co²⁺, and enhanced by Ba²⁺. PSA belonged to the EDTA-sensitive α-amylase. The purified enzyme showed high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), and oxidizing agents, such as sodium per borate and H₂O₂. In addition, PSA showed excellent compatibility with a wide range of commercial solid and liquid detergents at 30°C, suggesting potential application in the detergent industry. Maltotetraose was the specific end product obtained after hydrolysis of starch by the enzyme for an extended period of time, and was not further degraded.     

Pseudomonas aeruginosa Cif defines a distinct class of α/β epoxide hydrolases utilizing a His/Tyr ring-opening pair

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that secretes a multitude of virulence factors during the course of infection. Among these is Cif, an epoxide hydrolase (EH) that reduces the functional localization of the cystic fibrosis transmembrane conductance regulator in epithelial cells. In addition to being the first reported EH virulence factor, Cif possesses unique sequence deviations from canonical EH motifs. Foremost among these is the substitution of a histidine for the first epoxide ring-opening tyrosine in the active site. To test the functional equivalence of Tyr and His side chains at this position, we have generated the mutant Cif-H177Y. Structural analysis confirms that both the WT His and mutant Tyr side chains can be accommodated without large-scale conformational changes. However, the Tyr mutant is functionally inactive. Based on a detailed analysis of the structure of the Tyr mutant, it appears that Cif's main-chain conformation imposes a functional requirement for a His at this position. Comparison with canonical EH structures reveals additional conformational differences, which are coupled to divergent sequence characteristics. When used to probe the genomes of other opportunistic pathogens, these sequence-structure criteria uncover candidate sequences that appear to form a distinct subfamily of Cif-like epoxide hydrolases characterized by a conserved His/Tyr ring-opening pair.     

The significance of calcium ions on Pseudomonas fluorescens biofilms – a structural and mechanical study

The purpose of this study was to investigate the effects of calcium ions on the structural and mechanical properties of Pseudomonas fluorescens biofilms grown for 48?h. Advanced investigative techniques such as confocal laser scanning microscopy and atomic force spectroscopy were employed to characterize biofilm structure as well as biofilm mechanical properties following growth at different calcium concentrations. The presence of calcium during biofilm development led to higher surface coverage with distinct structural phenotypes in the form of a granular and heterogeneous surface, compared with the smoother and homogeneous biofilm surface in the absence of calcium. The presence of calcium also increased the adhesive nature of the biofilm, while reducing its elastic properties. These results suggest that calcium ions could have a functional role in biofilm development and have practical implications, for example, in analysis of biofouling in membrane-based water-treatment processes such as nanofiltration or reverse osmosis where elevated calcium concentrations may occur at the solid–liquid interface.     

Induction of γ-aminobutyric acid plays a positive role to Arabidopsis resistance against Pseudomonas syringae

Gamma‐aminobutyric acid (GABA) is an important metabolite which functions in plant growth, development, and stress responses. However, its role in plant defense and how it is regulated are largely unknown. Here, we report a detailed analysis of GABA induction during the resistance response to Pseudomonas syringae in Arabidopsis thaliana. While searching for the mechanism underlying the pathogen‐responsive mitogen‐activated protein kinase (MPK)3/MPK6 signaling cascade in plant immunity, we found that activation of MPK3/MPK6 greatly induced GABA biosynthesis, which is dependent on the glutamate decarboxylase genes GAD1 and GAD4. Inoculation with Pseudomonas syringae pv tomato DC3000 (Pst) and Pst‐avrRpt2 expressing the avrRpt2 effector gene induced GAD1 and GAD4 gene expression and increased the levels of GABA. Genetic evidence revealed that GAD1, GAD2, and GAD4 play important roles in both GABA biosynthesis and plant resistance in response to Pst‐avrRpt2 infection. The gad1/2/4 triple and gad1/2/4/5 quadruple mutants, in which the GABA levels were extremely low, were more susceptible to both Pst and Pst‐avrRpt2. Functional loss of MPK3/MPK6, or their upstream MKK4/MKK5, or their downstream substrate WRKY33 suppressed the induction of GAD1 and GAD4 expression after Pst‐avrRpt2 treatment. Our findings shed light on both the regulation and role of GABA in the plant immunity to a bacterial pathogen.     

Structure of the Pyoverdin PVD 2908 – a new Pyoverdin from Pseudomonas sp. 2908

An unknown siderophore (pyoverdin) was isolated from the strain Pseudomonas sp. 2908. The structure of the pyoverdin – called PVD 2908 – was elucidated by spectroscopic methods and degradation studies. Some other siderophores were identified by LC/ESI-MS-screening based on the knowledge of PVD 2908.     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Does Streptococcus mitis,a neonatal oropharyngeal bacterium,influence the pathogenicity of Pseudomonas aeruginosa?

Microorganisms in a biofilm might promote or suppress each other. We previously found that Pseudomonas aeruginosa (P. aeruginosa) and the normal colonized bacteria in the oropharynx, Streptococcus mitis (S. mitis), were the most common bacteria in the biofilm found on newborns' endotracheal tubes. Here, we found that S. mitis enhanced the adhesion and biofilm formation of P. aeruginosa. Furthermore, it alleviated the immune response induced by P. aeruginosa. These findings remind us that we should not ignore the role of traditionally viewed non-pathogenic bacteria in biofilms and provide new insights into exploring bacterial interaction mechanisms in biofilm related infections.     

A Carbapenem-Resistant Pseudomonas aeruginosa Isolate Harboring Two Copies of blaIMP-34 Encoding a Metallo-β-Lactamase

A carbapenem-resistant strain of Pseudomonas aeruginosa, NCGM1984, was isolated in 2012 from a hospitalized patient in Japan. Immunochromatographic assay showed that the isolate was positive for IMP-type metallo-β-lactamase. Complete genome sequencing revealed that NCGM1984 harbored two copies of bla_IMP-34, located at different sites on the chromosome. Each bla_IMP-34 was present in the same structures of the class 1 integrons, tnpA(ISPa7)-intI1-qacG-bla_IMP-34-aac(6'')-Ib-qacEdelta1-sul1-orf5-tniBdelta-tniA. The isolate belonged to multilocus sequence typing ST235, one of the international high-risk clones. IMP-34, with an amino acid substitution (Glu126Gly) compared with IMP-1, hydrolyzed all β-lactamases tested except aztreonam, and its catalytic activities were similar to IMP-1. This is the first report of a clinical isolate of an IMP-34-producing P. aeruginosa harboring two copies of bla_IMP-34 on its chromosome.