Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomonas aeruginosa

The cytochrome c-551 gene from Pseudomonas aeruginosa was cloned by using two oligonucleotide probes, which had been synthesized based on the known primary structure of the protein. The restriction map of the cloned DNA and sequence analysis showed that the cytochrome c-551 gene is located 50 bp downstream of the nitrite reductase gene, which has recently been cloned and sequenced. DNA sequence analysis also indicated that cytochrome c-551 is synthesized in vivo as a precursor having an amino-terminal signal sequence consisting of 22 amino acid residues.     

Two c-type cytochromes, NirM and NirC, encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors for nitrite reductase.

Three c-type cytochromes, NirM, NirC, and NirN, are encoded in the nirSMCFDLGHJEN gene cluster for cytochrome cd(1)-type nitrite reductase (NIR) of Pseudomonas aeruginosa. nirS is the structural gene for NIR. NirM (cytochrome c(551)) is reported to be a physiological electron donor for nitrite reductase. The respective functions of NirC and NirN have remained unclear. In this study, we produced recombinant NirC and NirN in P. aeruginosa, and purified them from the periplasmic fraction. N-terminal amino acid sequences of the purified proteins showed that the N-terminal 31 and 18 residues of NirC and NirN precursors were cleaved, respectively, indicating that cleaved peptides act as signals for membrane translocation. In addition, the ability of NirC for electron donation to nitrite reductase was investigated. NirC, as well as NirM, was able to mediate the electron donation from the membrane electron pathway to NIR, suggesting that the structural gene for NIR is followed by the genes for two electron donors for NIR.     

Molecular analysis of the folC gene of Pseudomonas aeruginosa

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase-dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co-transcribed.     

Isolation and Characterization of the algD gene of Pseudomonas syringae pv. phaseolicola and its distribution among other Pseudomonads and related Organisms

The gene coding for GDP-mannose dehydrogenase ( algD ) was isolated from a Pseudomonas syringae pv. phaseolicola genomic library using a polymerase chain reaction-generated heterologous DNA-probe from Pseudomonas aeruginosa . A total of 2123 base pairs were sequenced (accession number AF001555) and analysed for homologies to the alginate gene cluster of P. aeruginosa . Downstream from algD an alg8 homologue was found suggesting a similar arrangement of the alginate gene cluster in P. syringae pv. phaseolicola to that in P. aeruginosa . Also, the deduced amino acid sequence of algD shows high similarity to that of P. aeruginosa (0.9) and Azotobacter vinelandii (0.88). Southern hybridization experiments revealed that algD is widely distributed among members of the Pseudomonas rRNA homology group I. Among others, sequences homologous to algD were detected in the P. syringae pathovars lachrymans , mori , morsprunorum, pisi , savastanoi, tabaci and tomato as well as in Pseudomonas amygdali . For most of the algD positive organisms synthesis of alginate has been reported by other studies. However, algD homologues were also detected for the species Pseudomonas corrugata , Pseudomonas marginalis and Pseudomonas avenae ( Acidovorax avenae ), for which alginate biosynthesis has not yet been reported.     

Electron transfer between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa

The electron transfer equilibrium and kinetics between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa have been studied. The equilibrium constant K = ([Cyt(III)] . [Az(I)])/([Cyt(II)] . [Az(II))]) = 0.5 at 25 degrees C is about seven times smaller than that observed between the cytochrome c551 and the titrations confirmed a 43-mV difference between the mid-point potentials of +266 mV and +309 mV for the Alcaligenes and Pseudomonas azurins respectively. The kinetics of the reaction between Alcaligenes azurin and Pseudomonas cytochrome c551 were investigated by the temperature-jump chemical relaxation method. Only a single relaxation mode was observed throughout the range of concentrations and temperatures examined. Thus, the slow relaxation time observed in the reaction between P. aeruginosa azurin and cytochrome c551 is not observed with the Alcaligenes azurin. The simplest mechanism that can therefore be ascribed to the investigated system is: [formula: see text]. This scheme is similar to that proposed earlier for the reaction between P. aeruginosa azurin and cytochrome c551 but does not involve the conformational transition proposed for azurin. The specific rates for the electron transfer are still fast: 1.8 x 10(6) M-1 . s-1 and 3.0 x 10(6) M-1 . s-1 respectively at 25 degrees C.     

Sequence analysis of a gene product synthesized by Xanthomonas sp. during growth on natural rubber latex

Xanthomonas sp. secretes an extracellular protein (Mr approximately 70+/-5 kDa) during growth on purified natural rubber [poly(1,4-cis-isoprene)] but not during growth on water-soluble carbon sources such as glucose or gluconate. A 1.3 kbp DNA fragment coding for an internal part of the structural gene of the 70 kDa protein was amplified by nested polymerase chain reaction (PCR) using amino acid sequence information obtained after Edman degradation of selected trypsin-generated peptides of the purified 70 kDa protein. The PCR product was used as a DNA probe to clone the complete structural gene from genomic DNA of Xanthomonas sp. The sequenced DNA contained a 2037 bp open reading frame which coded for a polypeptide of 678 amino acids (Mr 74.6 kDa) and which included the features of the N-terminal signal peptidase cleavage site (Mr approximately 72.9 kDa for the mature protein). Analysis of the amino acid sequence revealed the presence of two heme binding motifs (CXXCH) and a approximately 20 amino acids long sequence that is conserved in the Paracoccus denitrificans and Pseudomonas aeruginosa diheme cytochrome c peroxidases (CCPs). This region includes a histidine residue (H519 in Xanthomonas sp. and H265 and H271 in the Pseudomonas strains, respectively) that is essential for activity in CCPs and that is also conserved in other bacterial oxidases. Blast analysis confirmed the relatedness of the 70 kDa protein to heme-containing oxidases and suggested that it is a member of a new family of relatively large (approximately 500 to approximately 1000 amino acids) extracellular proteins with so far unknown function being only far related in amino acid sequence to P. denitrificans and P. aeruginosa CCPs.     

The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase.

Interspecific complementation of a Xanthomonas campestris pv. campestris phosphomannose isomerase (PMI) mutant was used to isolate a cosmid from a genomic library of Rhizobium meliloti 2011 carrying the pmi gene of this strain. Subcloning experiments localized the coding region to a 2.0-kb SalI-ClaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames (ORFs), coding for 18- and 43-kDa polypeptides. The analysis of the gene function by gene disruption experiments showed that ORF2 codes for pmi. A comparison of the deduced amino acid sequence with the corresponding sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs revealed no significant homology, indicating that the isolated gene encodes a new type of PMI. The construction of a pmi-deficient mutant of R. meliloti using the sacB-sacR cassette technique showed that the loss of PMI activity does not affect the symbiotic properties of this strain.     

Selected mutations in a mesophilic cytochrome c confer the stability of a thermophilic counterpart

Mesophilic cytochrome c(551) of Pseudomonas aeruginosa (PA c(551)) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c(552) through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c(551) could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c(552), there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.     

The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.     

Electron self-exchange in Pseudomonas cytochromes

Electron self-exchange has been measured by an NMR technique for cytochromes c551 from Pseudomonas aeruginosa and Pseudomonas stutzeri. The rate for P. aeruginosa cyt c551 is 1.2 x 10(7) M-1 s-1 at 40 degrees C in 50 mM phosphate at pH 7. For P. stutzeri, under the same conditions, the rate is 4 x 10(7) M-1 s-1. For both cytochromes, the rate was independent of ionic strength up to 0.5 M in added NaC1, the enthalpy of activation was 20 +/- 4 kcal mol-1, and the entropy of activation was 38 +/- 10 cal mol-1 deg-1.     

A purification procedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa.

The production of the soluble cytochrome oxidase/nitrite reductase in the bacterium Pseudomonas aeruginosa is favoured by anaerobic conditions and the presence of KNO3(20g/l) in the culture medium. Of three methods commonly used for the disruption of bacterial suspensions (ultrasonication, liquid-shear homogenization and glass-bead grinding), sonication proved the most efficient in releasing the Pseudomonas cytochrome oxidase. A polarographic assay of Pseudomonas cytochrome oxidase activity with sodium ascorbate as substrate and NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride as electron mediator is described. A purification procedure was developed which can be used on the small scale (40-litre cultures) or the large scale (400-litre cultures) and provides high yields of three respiratory-chain proteins, Pseudomonas cytochrome oxidase, cytochrome c551 and azurin, in a pure state. A typical preparation of 250g of Ps.aeruginosa cell paste yielded 180mg of Pseudomonas cytochrome oxidase, 81 mg of Pseudomonas cytochrome c551 and 275mg of Pseudomonas azurin.     

Stabilization mechanism of cytochrome c552 from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus

Cytochrome c(552) (PH c(552)) from moderately thermophilic Hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome c(552) (HT c(552)) from thermophilic Hydrogenobacter thermophilus and cytochrome c(551) (PA c(551)) from mesophilic Pseudomonas aeruginosa. To understand the mechanism of stabilization of PH c(552), we introduced mutations into PH c(552) at five sites, which, in HT c(552), are occupied by the amino acids responsible for stability higher than the less stable PA c(551). When PH c(552) Val-78 was mutated to Ile, as found in HT c(552), the resulting variant showed increased stability. Mutation of Ala-7, Met-13, and Tyr-34 to the corresponding residues in PA c(551) (Phe, Val, and Phe, respectively) resulted in destabilization. We also found that PH c(552) Lys-43 contributed to stability through the formation of an attractive electrostatic interaction with Asp-39. These results suggest that the intermediate stability of PH c(552) is due to the amino acids at these five sites.     

Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics.     

Conferment of folding ability to a naturally unfolded apocytochrome c through introduction of hydrophobic amino acid residues

Hyperthermophilic Aquifex aeolicus cytochrome c(555) (AA c(555)) exceptionally folds even in the apo state, unlike general cytochromes c including mesophilic Pseudomonas aeruginosa cytochrome c(551) (PA c(551)), which is structurally homologous to AA c(555) in the holo state. Here we hypothesized that the exceptional apo AA c(555) folding can be attributed to nine hydrophobic amino acid residues and proved this using a PA c(551) variant (denoted as PA-nh) carrying the nine hydrophobic residues at structurally corresponding positions. Circular dichroism experiments showed that the apo PA-nh variant became folded, unlike the wild-type apo PA c(551), and exhibited much higher stability than the wild type. Another difference between the holo forms of AA c(555) and PA c(551) is the existence of an extra helix in the former. Introduction of the amino acid residues forming the extra helix of AA c(555) into the PA-nh variant did not significantly affect its folding ability in the apo state. Therefore, the nine hydrophobic residues introduced into the apo PA-nh variant were enough to confer the folding ability. PA c(551) represents the first example of the conversion of an intrinsically unfolded apocytochrome c into an autonomously folded one, which was revealed by means of a protein engineering method without heme. Although heme is generally considered to be a trigger of apocytochrome c folding, the present results demonstrate a new heme-independent folding mechanism.     

Nuclear-magnetic-resonance studies of Pseudomonas aeruginosa cytochrome c-551.

Nuclear magnetic resonance (NMR) spectroscopy was used to study Pseudomonas aeruginosa cytochrome c-551. Assignments of resonances to specific residues have been made. A low-resolution X-ray structure was used to aid assignments. A structural comparison was made between P. aeruginosa cytochrome c-551 and mammalian cytochrome c, based on comparisons of NMR data.     

Modification of the electron-transfer sites of Pseudomonas aeruginosa azurin by site-directed mutagenesis

Site-directed mutagenesis of the structural gene for azurin from Pseudomonas aeruginosa has been used to prepare azurins in which amino acid residues in two separate electron-transfer sites have been changed: His-35-Lys and Glu-91-Gln at one site and Phe-114-Ala at the other. The charge-transfer band and the EPR spectrum are the same as in the wild-type protein in the first two mutants, whereas in the Phe-114-Ala azurin, the optical band is shifted downwards by 7 nm and the copper hyperfine splitting is decreased by 4.10(-4)/cm. This protein also shows an increase of 20-40 mV in the reduction potential compared to the other azurins. The potentials of all four azurins decrease with increasing pH in phosphate but not in zwitterionic buffers with high ionic strength. The rate constant for electron exchange with cytochrome c551 is unchanged compared to the wild-type protein in the Phe-114-Ala azurin, but is increased in the other two mutant proteins. The results suggest that Glu-91 is not important for the interaction with cytochrome c551 and that His-35 plays no critical role in the electron transfer to the copper site.     

Isolation and characterization of the gene from Pseudomonas syringae pv. phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase

Summary The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) fromPseudomonas syringae pv.phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated M, of 36520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by theP. aeruginosa argF and theEscherichia coli argI andargF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be namedargK.     

Stabilization of Pseudomonas aeruginosa cytochrome c(551) by systematic amino acid substitutions based on the structure of thermophilic Hydrogenobacter thermophilus cytochrome c(552)

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c(551) (PA c(551)) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c(551) with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(551) with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c(551) with HT c(552). In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c(552). We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c(551) can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c(552). The higher stability of HT c(552) may in part be attributed to some of these substitutions.