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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.

Alcaligenes↗

Peritoneal dialysis-associated peritonitis caused by Alcaligenes xylosoxidans.

Despite significant progress to decrease its incidence, peritonitis remains the main source of morbidity and treatment failure in patients on continuous ambulatory peritoneal dialysis (CAPD). The majority of cases of peritonitis result from infection with aerobic gram-positive (Staphylococcus epidermidis and Staphylococcus aureus), or gram-negative organisms. Less common organisms that are also reported include anaerobic bacteria, fungi, and mycobacteria, which collectively account for less than 10% of isolates cultured. We report a case of peritoneal dialysis-associated peritonitis, and review the literature on peritonitis caused by Alcaligenes species. Alcaligenes xylosoxidans is a nonfermenting gram-negative rod and opportunistic pathogen that is motile with peritrichous flagella. The clinical features and microbiological data of our case, as well as the other previously reported cases of peritonitis caused by Alcaligenes species show no particular pattern of peritoneal dialysate cell count. However, the rate of recurrence of peritonitis is characteristically high. The cause of such a high rate of recurrence of peritonitis is probably a reflection of the predilection of Alcaligenes species to cause infection in the 'sicker' patients, and the almost universal resistance of this species to most antimicrobial agents. We, therefore, recommend that catheter removal be undertaken as early as the identification of the organism is made. Whether patients should be allowed to return to CAPD after recovery is a more difficult question. We suggest that a reevaluation of the patient's overall status be undertaken, including personal hygiene, exchange technique, presence of diabetes mellitus, malnutrition, and/or other factors that may render the patient more prone to infection with opportunistic pathogens.

Alcaligenes↗

Treatment of dairy wastewater using a selected bacterial isolate, Alcaligenes sp. MMRR7.

Physicochemical and biologic analysis of dairy wastewater showed that the effluent had a high organic load (chemical oxygen demand [COD]: 5095 mg/L), an acidic pH (6.4), and a high probability of coliforms (most probable number [MPN] >1100). The various bacterial strains isolated and purified were identified as Sporolactobacillus sp., Citrobacter sp., Pseudomonas sp., Alcaligenes sp., Bacillus sp., Staphylococcus sp., and Proteus sp., as per the Bergey's manual of systematic bacteriology. Among the five selected bacterial strains, the strain designated as MMRR7 and identified as Alcaligenes sp. was found to give a maximum reduction in COD (62%) in 5 d of incubation. Chemical coagulation using alum at a concentration of 0.5 g/100 mL was found to be effective in the primary treatment of the effluent. Studies on free-cell treatment of the coagulated effluent with the selected bacterial strain Alcaligenes sp. MMRR7 gave a maximum COD reduction of 91% in 120 h. This study clearly indicates the possibility of using Alcaligenes sp. MMRR7 for the effective treatment of dairy wastewater.

Alcaligenes↗

An Alcaligenes faecalis isolate from turkeys: pathogenicity in selected avian and mammalian species.

An Alcaligenes faecalis isolate of known pathogenicity for turkeys was examined for adherence and cytotoxicity in tracheal organ cultures of turkeys, chickens, Japanese quail, guinea pigs, hamsters, and mice, and for colonization and pathogenicity in these 6 species. Adherence and colonization were detected by fluorescent antibody staining. Infected and noninfected tracheal rings were examined by phase-contrast microscopy for cytotoxicity (ciliostasis, blebing of the cell membrane, and sloughing of the ciliated epithelium). Alcaligenes faecalis adhered to the tracheal rings of all species examined. Cytotoxicity was apparent in the tracheal rings of turkeys, quail, and chickens. Cytotoxicity was not detected in tracheal rings from the mammalian species. Alcaligenes faecalis colonization of turbinates and tracheas of intact turkeys and quail was detected. Clinical signs of alcaligenes rhinotracheitis were observed and histopathologic characteristics of the disease were detected. Chickens, guinea pigs, hamsters, and mice were refractory to infection with this isolate of A faecalis.

Adhesiveness↗

Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22.

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (betadelta) and an NADH:acceptor oxidoreductase (alphagamma)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed.

Alcaligenes↗

Pyrimidine base and ribonucleoside utilization by the Pseudomonas alcaligenes group.

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of the Pseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth of Pseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supported Pseudomonas alcaligens ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by either P. pseudoalcaligenes or P. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts of P. pseudoalcaligenes and P. alcaligenes. In P. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.

Amidohydrolases↗

The phenotype enhancement method identifies the Xcp outer membrane secretion machinery from Pseudomonas alcaligenes as a bottleneck for lipase production.

Pseudomonas alcaligenes M-1 has been selected from an intensive screening for micro-organisms that can naturally produce a lipase active in detergent formulations. The lipase expression has been increased to allow high level secretion from Pseudomonas alcaligenes, via the introduction of multi-copy plasmids. In order to improve the lipase yield further, the phenotype enhancement method has been developed. This idea comprises the reintroduction of a cosmid library with random chromosomal fragments in a P. alcaligenes strain with already high lipase productivity. One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion genes. These xcp-genes are clustered in two divergently transcribed operons similar to the situation in Pseudomonas aeruginosa. Remarkably and dissimilar to P. aeruginosa, in between the two xcp gene clusters, two reading frames of unknown function--OrfV and OrfX--are present. For OrfX no equivalent can be found in the known protein data bases. On the other hand, OrfV shows homology to the regulatory proteins MalT and AcoK. Some evidence is provided that suggests that OrfV acts as a regulator of the xcp operons. A model is proposed for the regulation of the secretion system from P. alcaligenes.

Amino Acid Sequence↗

Three-dimensional structure of cytochrome c' from two Alcaligenes species and the implications for four-helix bundle structures.

The three-dimensional structures of two cytochromes c' have been determined in order to analyse the common features of proteins of this family and their relationship with other four-helix bundle structures. The structure of cytochrome c' from Alcaligenes sp was determined by molecular replacement supplemented with the iron anomalous scattering and the use of a single isomorphous heavy-atom derivative, and was refined using synchrotron data to 1.8 A resolution. The final model, comprising 956 protein atoms (one monomer) and 89 water molecules, has a final R value of 0.188 for all data in the range 20.0-1.8 A resolution (14 673 reflections). The structure of the cytochrome c' from Alcaligenes denitrificans is isomorphous and essentially identical (r.m.s. deviation for all atoms 0.36 A). Although its amino-acid sequence has not been determined chemically, only four differences from that of Alcaligenes sp cytochrome c' were identified by the X-ray analysis. The final model for Alcaligenes denitrificans cytochrome c', comprising 953 protein atoms and 75 water molecules, gave a final R factor of 0.167 for all data in the range 20.0-2.15 A (8220 reflections). The cytochrome c' monomer forms a classic four-helix bundle, determined by the packing of hydrophobic side chains around the enclosed haem group. There are very few cross-linking hydrogen bonds between the helices, the principal side-chain hydrogen bonding involving one of the haem propionates and a conserved Arg residue. The cytochrome c' dimer is created by a crystallographic twofold axis. Monomer-monomer contacts primarily involve the two A helices, with size complementarity of side chains in a central solvent-excluded portion of the interface and hydrogen bonding at the periphery. Both species have a pyroglutamic acid N-terminal residue. The haem iron is five-coordinate, 0.32 A out of the haem plane towards the fifth ligand, His120. The unusual magnetic properties of the Fe atom may be linked to a conserved basic residue, Arg124, adjacent to His120.

Journal Article↗

Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9.

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.

Blotting, Southern↗

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

Bacterial Proteins↗

[Alcaligenes xylosoxidans--a pathogen of chronic ear infection].

INTRODUCTION: We discuss a case of a 48-year old man admitted to our Department with symptoms of chronic ear infection, that didn't answer to typical conservative treatment. The culture of the discharge was positive for Alcaligenes xylosoxidans. MATERIAL AND METHODS: We report characteristic features of that bacteria and we compare them with features of other pathogens typical for otitis media. We present performed treatment too. RESULTS: Alcaligenes xylosoxidans is a nonfermenting gram-negative rod. It can be confused with other nonfermentive, gram-negative rods, especially Pseudomonas species, in clinical specimens so that its role as a significant pathogen may be underestimated. Alcaligenes xylosoxidans is a part of normal human flora, especially skin and gastrointestinal tract, and can cause serious infections in human (especially in patients with immunodeficiency and premature children). CONCLUSIONS: Reports about Alcaligenes xylosoxidans as a cause of chronic otitis media are very rare and it can be supposed to stay a new kind of ear infection.

Achromobacter denitrificans↗

Bacteremia caused by Achromobacter and Alcaligenes species in 46 patients with cancer (1989-2003).

BACKGROUND: Achromobacter and Alcaligenes are emerging infectious gram-negative bacterial species that can affect immunosuppressed patients. The authors sought to determine the incidence and characteristics of bloodstream infections caused by these organisms in patients with underlying malignancies. METHODS: All consecutive episodes of hematogenous Achromobacter and Alcaligenes infections recorded from December 26, 1989, to July 27, 2003, were studied retrospectively. RESULTS: Fifty-two episodes occurred in 46 patients; 31 patients (67%) had hematologic malignancies, and 24 (52%) experienced neutropenia (< 500 cells/microL). Diabetes mellitus was present in 12 patients (26%), and high-dose corticosteroids were administered to 12 patients (26%). Seventeen of the 52 infectious episodes (33%) were nosocomial in origin, and 10 patients (22%) had sepsis syndrome. Achromobacter xylosoxidans was the most common cause of infection (47 of 52 episodes [94%]), followed by Ach. denitrificans (2 of 52 episodes [4%]) and Alcaligenes faecalis (1 of 52 episodes [2%]). Twenty-seven episodes (52%) were polymicrobial, and 3 patients (7%) had concurrent fungemia. Infected intravascular catheters were present in 13 of 52 cases (25%), pneumonia was encountered in 6 of 52 cases (12%), and urinary tract infections were present in 5 of 52 cases (10%). Most isolates exhibited in vitro susceptibility to carbapenems, antipseudomonal penicillins, and trimethoprim-sulfamethoxazole. Resistance to ciprofloxacin, levofloxacin, aminoglycosides, and monobactam was common. Seven deaths (15%) were attributable to Achromobacter species. Incidence rates for sepsis syndrome, multiorgan dysfunction (Acute Physiology and Chronic Health Evaluation [APACHE] II score > 16), and use of mechanical ventilation and pressor support were significantly higher in patients who died (P < 0.001). Logistic regression analysis revealed that sepsis syndrome and high APACHE II scores were predictors of increased 30-day mortality. CONCLUSIONS: Most infections caused by this group of nonfermentative gram-negative bacteria were attributable to Ach. xylosoxidans, and only one-third were acquired during hospitalization. The presence of sepsis syndrome has evolved as an independent predictor of poor outcome in patients with high-risk malignancies accompanied by Achromobacter bloodstream infections.

Achromobacter↗

Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867.

Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized. A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. The proteome profiles of P. alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis. Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing. Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms. The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp. U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P. alcaligenes. In addition, the induction of stress proteins and other adaptation phenomena were also observed.

Bacterial Proteins↗

Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867.

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) utilizes the gentisate pathway for the degradation of aromatic hydrocarbons. The gene encoding the alternative sigma (sigma) factor sigma(54), rpoN, was cloned from strain P25X and a rpoN knock-out strain, designated G54, was constructed by insertional inactivation with a kanamycin resistance gene cassette. The role of sigma(54) in the physiological response of P. alcaligenes P25X to gentisate induction was assessed by comparing the global protein expression profiles of the wild-type P25X with the rpoN mutant strain G54. Analysis of two-dimensional polyacrylamide gel electrophoresis gels showed that 39 out of 355 prominent protein spots exhibited differential expression as a result of the insertional inactivation of rpoN. Identification of the protein spots by matrix-assisted laser desorption/ionization-time of flight/time of flight revealed a wide diversity of proteins that are affected by the sigma(54) mutation, the largest group being proteins that are involved in carbon metabolism. The strictly inducible gentisate 1,2-dioxygenase, one of two isofunctional copies of the key enzyme in the gentisate pathway, and enzymes of the TCA cycle, pyruvate metabolism and gluconeogenesis were part of this group. Other proteins that are part of the sigma(54) regulon include enzymes implicated in nitrogen metabolism, transport proteins, stress-response proteins and proteins involved in cell motility. The results of this study showed that sigma(54) plays a global regulatory role in the expression of a wide variety of genes in P. alcaligenes, including the wild-type response to the presence of the aromatic inducer, gentisate.

Electrophoresis, Gel, Two-Dimensional↗

Catabolic instability, plasmid gene deletion and recombination in Alcaligenes sp. BR60.

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR 40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10(-10) cell-1 generation-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BglII, HindIII and SalI restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R68.45 into Alcaligenes sp. BR 60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BglII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba+ mutants of Alcaligenes sp. BR60.

Alcaligenes↗

Transfer and expression of PCB-degradative genes into heavy metal resistant Alcaligenes eutrophus strains.

Sites polluted with organic compounds frequently contain inorganic pollutants such as heavy metals. The latter might inhibit the biodegradation of the organics and impair bioremediation. Chromosomally located polychlorinated biphenyl (PCB) catabolic genes of Alcaligenes eutrophus A5, Achromobacter sp. LBS1C1 and Alcaligenes denitrificans JB1 were introduced into the heavy metal resistant Alcaligenes eutrophus strain CH34 and related strains by means of natural conjugation. Mobile elements containing the PCB catabolic genes were transferred from A. eutrophus A5 and Achromobacter sp. LB51C1 into A. eutrophus CH34 after transposition onto their endogenous IncP plasmids pSS50 and pSS60, respectively. The PCB catabolic genes of A. denitrificans JB1 were transferred into A. eutrophus CH34 by means of RP4::Mu3A mediated prime plasmid formation. The A. eutrophus CH34 transconjugant strains expressed both catabolic and metal resistance markers. Such constructs may be useful for the decontamination of sites polluted by both organics and heavy metals.

Alcaligenes↗

Molecular epidemiology of Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans in a cystic fibrosis center.

Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans have been isolated with increasing frequency from the sputum of patients with cystic fibrosis in a pediatric hospital. In 1994-95, 27 of 120 patients were persistently colonized, 17 with Burkholderia cepacia, eight with Alcaligenes xylosoxidans, and five with Stenotrophomonas maltophilia. Genotyping of 220 clinical isolates revealed that most of the Burkholderia cepacia strains were clonally related, suggesting either cross-infection or a common source of exposure. In contrast, neither cross-infection nor a common source of exposure appear to have occurred in the cases of Alcaligenes xylosoxidans or Stenotrophomonas maltophilia.

Alcaligenes↗

Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor.

A Gram (-) coccobacillary bacterium, J(T), was isolated from a graywater bioprocessor. 16S rRNA and biochemical analysis has revealed strain J(T) closely resembles Alcaligenes faecalis ATCC 8750T and A. faecalis subsp. parafaecalis DSM 13975T, but is a distinct, previously uncharacterized isolate. Strain J(T), along with the type strain of A. faecalis and its previously described subspecies share the ability to aerobically degrade phenol. The degradation rates of phenol for strain J(T) and reference phenol degrading bacteria were determined by photometrically measuring the change in optical density when grown on 0.1% phenol as the sole carbon source, followed by addition of Gibb's reagent to measure depletion of substrate. The phenol degradation rates of strain J(T) was found to exceed that of the phenol hydroxylase group III bacterium Pseudomonas pseudoalcaligenes, with isolate J(T) exhibiting a doubling time of 4.5 h. The presence of the large subunit of the multicomponent phenol hydroxylase gene in strain J(T) was confirmed by PCR. The presence of the nirK nitrite reductase gene as demonstrated by PCR as well as results obtained from nitrite media indicated denitrification at least to N2O. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA DNA hybridization, we propose assigning a novel subspecies of Alcaligenes faecalis, to be named Alcaligenes faecalis subsp. phenolicus with the type strain J(T) (= DSM 16503) (= NRRL B-41076).

Alcaligenes faecalis↗