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PubMed1983

Pathogenesis of change in the upper respiratory tracts of turkeys experimentally infected with an Alcaligenes faecalis isolate.

The course of changes within the upper respiratory tracts of turkey poults experimentally infected with Alcaligenes faecalis was studied. The initial change observed (5 days post-inoculation) was colonization of the upper respiratory tract by the bacterium. Changes in the nasal turbinates and trachea were first apparent as a focal loss of cilia but subsequently developed into a general loss of cilia (11 days post-inoculation). Eventually, the entire ciliated epithelial layer in the cranial region of the trachea was lost (13 days post-inoculation). With the loss of cilia and ciliated cells, a highly viscous mucus was able to accumulate in the anterior one-half to two-thirds of the trachea. In addition, changes in the gross structure of the trachea (flaccid trachea) were observed in all poults inoculated with A. faecalis. There was an apparent gradation in the severity of these changes from severe in the cranial region of the trachea to mild in the region just anterior to the bronchial bifurcation. The observations resulting from A. faecalis infection indicated two major tracheal changes responsible for the chronic and sometimes severe nature of this disease. These changes included a loss of ciliary activity and a flaccid trachea which together resulted in the accumulation and stasis of mucus and tracheal collapse.

Alcaligenes↗
PubMed1969

Purification and characterization of beta-glucosidase of Alcaligenes faecalis.

A cellobiose-utilizing bacterium isolated from sugar cane bagasse and identified as a strain of Alcaligenes faecalis (ATCC 21400) produced an inducible beta-glucoside-splitting enzyme. The enzyme was purified by a series of streptomycin and ammonium sulfate fractionations and by Sephadex and diethylaminoethyl column chromatography. The final preparation was purified 130-fold, with a recovery of about 10% of the initial enzyme activity. The enzyme had a wide pH range, with optimal activity at pH 6.0 to 7.0. The enzyme was stable in solution at pH 6.5 to 7.8 when kept at 30 C for 2 hr, but it was destroyed by temperatures above 55 C. At 58 and 60 C, the time required to inactivate 90% of the initial activity was 16 and 6.5 min, respectively. An activation energy of 9,500 cal/mole and a K(m) of 1.25 x 10(-4)m were obtained by using p-nitrophenyl beta-glucoside as a substrate. The K(i) value and hydrolysis of cellobiose by the enzyme indicated a high affinity of the enzyme for the cellobiose. The enzyme had its specificity on beta-glucosidic linkage and the rate of hydrolisis of glucosides depended upon the nature of the aglycon moiety. The inactivation studies showed the presence of sulfhydryl groups in the enzyme. The activity of the enzyme was easily destroyed by the Cu(++) and Hg(++) ions. The Michaelis-Menton relationship and the rate of heat inactivation indicated the presence of one type of noninteracting active site in the bacterial beta-glucosidase. Molecular weight of the enzyme was estimated by gel filtration (Sephadex G-200) and sucrose density gradient, and a value of 120,000 to 160,000 was obtained.

Alcaligenes↗
PubMed1971

Dissimilation of aromatic compounds by Alcaligenes eutrophus.

The range of aromatic compounds that support the growth of Alcaligenes eutrophus has been determined, and the pathways used for the dissimilation of these substrates have been explored, largely by enzymatic analyses. The beta-ketoadipate pathway operates in the dissimilation of benzoate and p-hydroxybenzoate; the genetisate pathway, in the dissimilation of m-hydroxybenzoate; and the meta cleavage pathway, in the dissimilation of phenol and p-cresol. l-Tryptophan is oxidized via anthranilate; but the metabolic fate of anthranilate was not established. The metabolism of the three stereoisomers of muconic acid was also examined.

Adipates↗
PubMed1971

Regulation of the -ketoadipate pathway in Alcaligenes eutrophus.

The regulation of the synthesis of the inducible enzymes that mediate the reactions of the beta-ketoadipate pathway in Alcaligenes eutrophus has been examined by determining the inductive responses of the wild type and of mutants derived from it to metabolites of the pathway. The system of control differs in many respects from those which operate in the genera Pseudomonas and Acinetobacter.

Adipates↗
PubMed1973

Evidence for the existence of an aerobic pathway for synthesis of monounsaturated fatty acids by Alcaligenes faecalis.

Studies on the composition of total fatty acids of Alcaligenes faecalis harvested at different growth phases have been carried out. Ability of the organism to desaturate palmitic and stearic acid has also been tested. The organism contained palmitic (16:0), stearic (18:0), palmitoleic (16:1), cis-vaccenic (18:1), cyclopropane (17: big dn tri, open and 19: big dn tri, open), and three hydroxy acids. Increase in cyclopropane acids and corresponding decrease in monounsaturated acids in direct proportion to the age of the culture were observed, whereas other fatty acids remained relatively unaltered. A growing culture of the organism was found to desaturate [1-(14)C]palmitic acid supplied in the medium to hexadecanoic acid. Resting cells desaturated [1-(14)C]palmitic and [1-(14)C]stearic acid giving rise to about 50% of (14)C in the COOH group of corresponding monounsaturated fatty acids.

Acetates↗
PubMed1976

Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.

Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.

Alcaligenes↗
PubMed1976

Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus.

Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies. Chorismate mutase was feedback inhibited by phenylalanine. In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM. When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred. The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity. Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity. The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C. Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine. Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors. Tryptophan inhibited the enzyme competitively. Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM). The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C. Prephenate dehydrogenase was feedback inhibited by tyrosine. Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide. The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM). Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM. The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C. It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production.

Alcaligenes↗
PubMed1978

Metabolism of cyclohexane carboxylic acid by Alcaligenes strain W1.

Thirty-three microorganisms capable of growth with cyclohexane carboxylate as the sole source of carbon were isolated from mud, water, and soil samples from the Aberystwyth area. Preliminary screening and whole-cell oxidation studies suggested that, with one exception, all of the strains metabolized the growth substrate by beta-oxidation of the coenzyme A ester. This single distinctive strain, able to oxidize rapidly trans-4-hydroxycyclohexane carboxylate, 4-ketocyclohexane carboxylate, p-hydroxybenzoate, and protocatechuate when grown with cyclohexane carboxylate, was classified as a strain of Alcaligenes and given the number W1. Enzymes capable of converting cyclohexane carboxylate to p-hydroxybenzoate were induced by growth with the alicyclic acid and included the first unambiguous specimen of a cyclohexane carboxylate hydroxylase. Because it is a very fragile protein, attempts to stabilize the cyclohexane carboxylate hydroxylase so that a purification procedure could be developed have consistently failed. In limited studies with crude cell extracts, we found that hydroxylation occurred at the 4 position, probably yielding the trans isomer of 4-hydroxycyclohexane carboxylate. Simultaneous measurement of oxygen consumption and reduced nicotinamide adenine dinucleotide oxidation, coupled with an assessment of reactant stoichiometry, showed the enzyme to be a mixed-function oxygenase. Mass spectral analysis enabled the conversion of cyclohexane carboxylate to p-hydroxybenzoate by cell extracts to be established unequivocally, and all of our data were consistent with the pathway: cyclohexane carboxylate --> trans-4-hydroxycyclohexane carboxylate --> 4-ketocyclohexane carboxylate --> p-hydroxybenzoate. The further metabolism of p-hydroxybenzoate proceeded by meta fission and by the oxidative branch of the 2-hydroxy-4-carboxymuconic semialde-hyde-cleaving pathway.

Alcaligenes↗
PubMed1981

Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus.

The nickel-dependent chemolithoautotrophic growth of Alcaligenes eutrophus is apparently due to a requirement of nickel for active hydrogenase formation. Cells grown heterotrophically with fructose and glycerol revealed a specific activity of soluble and membrane-bound hydrogenase which was severalfold higher than the normal autotrophic level. The omission of nickel from the medium did not affect heterotrophic growth, but the soluble hydrogenase activity was reduced significantly. In the presence of ethylenediaminetetraacetic acid (EDTA), almost no hydrogenase activity was detected. The addition of nickel allowed active hydrogenase formation even when EDTA was present. When chloramphenicol was added simultaneously with nickel to an EDTA-containing medium, almost no hydrogenase activity was found. This indicates that nickel ions are involved in a process which requires protein synthesis and not the direct reactivation of a preformed inactive protein. The formation of the membrane-bound hydrogenase also appeared to be nickel dependent. Autotrophic CO2 assimilation did not specifically require nickel ions, since formate was utilized in the presence of EDTA and the activity of ribulosebisphosphate carboxylase was not affected under these conditions.

Alcaligenes↗
PubMed1981

Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus.

Mutants defective in chemolithoautotrophic growth (Aut-) have been isolated from Alcaligenes eutrophus strains H16, N9A, G27, and TF93. Spontaneous Aut- mutants were obtained only with strain TF93. Mutants of the other strains were selected after conventional mutagenesis or treatment with mitomycin. Most of the mutants, including the spontaneous Aut- strains, lacked hydrogenase activity (Hox-) but possessed the ability to fix carbon dioxide (Cfx+). Agar mating of A. eutrophus H16 with Hox- mutants of the various strains resulted in transconjugants which had recovered the ability to grow autotrophically and to express activity of hydrogenase as examined by enzymatic and immunochemical analysis. Transfer of hydrogen-oxidizing ability occurred in the absence of a mobilizing plasmid such as Rp4. The transfer frequency was particularly high (ca. 10(-2) per donor) when the spontaneous Hox- mutants of strain TF93 were used as recipients. These strains proved to be plasmid free, whereas donors, transconjugants, and the mutagen-treated Hox- mutants contained a large plasmid (molecular weight, 270 +/- 10 X 10(6) revealed by agarose gel electrophoresis. The results allow the conclusion that A. eutrophus H16 harbors a self-transmissible plasmid designated pHG1, which carries information for hydrogen-oxidizing ability.

Alcaligenes↗
PubMed1982

Depression of hydrogenase during limitation of electron donors and derepression of ribulosebisphosphate carboxylase during carbon limitation of Alcaligenes eutrophus.

Alcaligenes eutrophus did not form the key enzymes of autotrophic metabolism, the soluble and particulate hydrogenases and ribulosebisphosphate carboxylase (RuBPC), during heterotrophic growth on succinate in batch cultures. During succinate-limited growth in a chemostat, high activities of both hydrogenases were observed. With decreasing dilution rate (D) the steady-state hydrogenase activity (H) followed first-order kinetics, expressed as follows: H = Hmax .e-alpha.D. An identical correlation was observed when autotrophic growth in a chemostat was limited by molecular hydrogen. During autotrophic growth under oxygen or carbon dioxide limitation, the activity if the soluble hydrogenase was low. These data suggested that hydrogenase formation depended on the availability of reducing equivalents to the cells. RuBPC activities were not correlated with the hydrogenase activities. During succinate-limited growth, RuBPC appeared at intermediate activities. During autotrophic growth in a carbon dioxide-limited chemostat, RuBPC was highly derepressed. RuBPC activity was not detected in cells that suffered from energy limitation with a surplus of carbon, as in a heterotrophic oxygen-limited chemostat, nor was it detected in cells limited in carbon and energy, as in the case of complete exhaustion of a heterotrophic substrate. From these data I concluded that RuBPC formation in A. eutrophus depends on two conditions, namely, carbon starvation and an excess of reducing equivalents.

Alcaligenes↗
PubMed1982

Effect of phosphoglycerate mutase deficiency on heterotrophic and autotrophic carbon metabolism of Alcaligenes eutrophus.

Mutants of Alcaligenes eutrophus were isolated on the basis of their inability to grow on succinate as the sole source of carbon and energy. The mutants also failed to grow on other gluconeogenic substrates, including pyruvate, acetate, and citrate. Simultaneously, they had lost their capability for autotrophic growth. The mutants grew, but slower than the wild type, on fructose or gluconate. Growth retardation on gluconate was more pronounced. The mutants lacked phosphoglycerate mutase activity, and spontaneous revertants of normal growth phenotype had regained the activity. The physiological characteristics of the mutants indicate the role of phosphoglycerate mutase in heterotrophic and autotrophic carbon metabolism of A. eutrophus. Although the enzyme is necessary for gluconeogenesis during heterotrophic growth on three- or four-carbon substrates, its glycolytic function is not essential for the catabolism of fructose or gluconate via the Entner-Doudoroff pathway. The enzyme is required during autotrophic growth as a catalyst in the biosynthetic route leading from glycerate 3-phosphate to pyruvate. It is suggested that the mutants accomplish the complete degradation of fructose and gluconate mutase lesion. The catabolically produced triose phosphates are converted to fructose 6-phosphate which is rechanneled into the Entner-Doudoroff pathway. This carbon recycling mechanism operates less effectively in mutant cells growing on gluconate.

Alcaligenes↗
PubMed1982

Nickel in the catalytically active hydrogenase of Alcaligenes eutrophus.

Nickel is a constituent of soluble and particulate hydrogenase of Alcaligenes eutrophus. Incorporation of 63Ni2+ revealed that almost the total nickel taken up by the cells was bound to the protein. Chromatography of a crude extract on diethylaminoethyl cellulose demonstrated an association of 63Ni2+ with soluble and particulate hydrogenase, supported by further analysis like polyacrylamide gel electrophoresis. Unspecific binding of 63Ni2+ to the protein was excluded by comparison with a mutant extract free of hydrogenase protein. X-ray fluorescence analysis of the homogeneous soluble hydrogenase indicated the presence of 2 mol of nickel per mol of enzyme, whereas the amount of nickel determined by incorporation of 63Ni2+ was calculated to be approximately 1 mol/mol of enzyme. Cells grown under nickel limitation contained catalytically inactive, but serologically active, soluble and particulate hydrogenase. The immunochemical reactions were only partially identical with the enzyme from nickel-cultivated cells indicating a structural modification of the proteins in the absence of nickel. It is concluded that nickel is essential for the catalytic activity of hydrogenase and not involved as a regulatory component in the synthesis of this enzyme.

Alcaligenes↗
PubMed1983

Regulation of hydrogenase formation is temperature sensitive and plasmid coded in Alcaligenes eutrophus.

Alcaligenes eutrophus grew well autotrophically with molecular hydrogen at 30 degrees C, but failed to grow at 37 degrees C (Hox Ts). At this temperature the strain grew well heterotrophically with a variety of organic compounds and with formate as an autotrophic substrate, restricting the thermolabile character to hydrogen metabolism. The soluble hydrogenase activity was stable at 37 degrees C. The catalytic properties of the wild-type enzyme were identical to those of a mutant able to grow lithoautotrophically at 37 degrees C (Hox Tr). Soluble hydrogenase was not rapidly degraded at elevated temperatures since the preformed enzyme remained stable for at least 5 h in resting cells or was diluted by growth, as shown in temperature shift experiments. Immunochemical studies revealed that the formation of the hydrogenase proteins was temperature sensitive. No cross-reactivity was detected above temperatures of 34 degrees C. The genetic information of Hox resides on a self-transmissible plasmid in A. eutrophus. Using Hox Tr mutants as donors of hydrogen-oxidizing ability resulted in Hox+ transconjugants which not only had recovered plasmid pHG1 and both hydrogenase activities but also were temperature resistant. This is evidence that the Hox Tr phenotype is coded by plasmid pHG1.

Alcaligenes↗
PubMed1983

Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus.

Alcaligenes eutrophus formed ribulosebisphosphate carboxylase (RuBPCase; EC 4.1.1.39) when grown on fructose. Addition of sodium fluoride (NaF) to fructose minimal medium resulted in a slightly decreased growth rate and a rapid fivefold increase in RuBPCase specific activity. With citrate, a glucogenic carbon source, RuBPCase was also formed, However, addition of NaF to cells growing on citrate resulted in a 50% decrease in RuBPCase specific activity. Among the enzymes of fructose catabolism, NaF (10 mM) inhibited enolase in vitro by 98% and gluconate 6-phosphate dehydratase by 87%. Inhibition of the dehydratase by NaF was insignificant in vivo, as determined with a mutant defective in phosphoglycerate mutase activity. Growth of this mutant on fructose was not inhibited by NaF, and only a minor increase in RuBPCase activity was observed. From these results, we concluded that the product of the enolase reaction, phosphoenolpyruvate, played a role in RuBPCase formation. Addition of H2 or formate to the wild type growing on fructose or citrate did not affect the growth rate but resulted in rapid formation of RuBPCase activity. Mutants impaired in H2 metabolism formed RuBPCase at a low rate during growth on fructose plus H2 but at a high rate on formate. Apparently, additional reductant from H2 or formate metabolism induced RuBPCase formation in A. eutrophus.

Alcaligenes↗
PubMed1983

Control of catechol meta-cleavage pathway in Alcaligenes eutrophus.

Alcaligenes eutrophus 335 (ATCC 17697) metabolizes phenol and p-cresol via a catechol meta-cleavage pathway. Studies with mutant strains, each defective in an enzyme of the pathway, showed that the six enzymes assayed are induced by the primary substrate. Studies with a putative polarity mutant defective in the expression of aldehyde dehydrogenase suggested that the structural genes encoding this and subsequent enzymes of the pathway exist in the same operon. From studies with mutant strains that constitutively synthesize catechol 2,3-oxygenase and subsequent enzymes and from the coordination of repression of these enzymes by p-toluate, benzoate, and acetate, it is proposed the catechol 2,3-oxygenase structural gene is situated in this operon (2,3-oxygenase operon). Studies with regulatory mutant strains suggest that the 2,3-oxygenase operon is under negative control.

Alcaligenes↗
PubMed1983

Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB113 (RP4::mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2.

Plasmid pULB113 (RP4::mini-Mu), which contains the mini-Mu transposon, promoted both homologous and heterologous gene transfer from Pseudomonas fluorescens 6.2 and Alcaligenes eutrophus CH34. Homologous gene transfer in P. fluorescens 6.2 and A. eutrophus CH34 occurred at a frequency of 10(-4) to 10(-5), and recombinants inherited unselected recessive markers, suggesting a process of chromosome mobilization. Loci involved in autotrophic growth were among those transferred in A. eutrophus. In heterospecific matings, markers were transferred from P. fluorescens to A. eutrophus, Salmonella typhimurium LT2, and Escherichia coli, from A. eutrophus to P. fluorescens, and from Erwinia carotovora subsp. chrysanthemi to A. eutrophus. Heterospecific matings resulted in the formation of R-prime plasmids at frequencies of 10(-7) to 10(-4) per transferred plasmid. When S. typhimurium was the recipient, we observed R-prime plasmids with both restriction-proficient and restriction-deficient strains, although restriction markedly affected the frequency of transfer of pULB113. R-prime plasmids were quite stable, but lost the transposed marker more easily in a rec+ background than in a recA background, suggesting excision of transposed material by reciprocal recombination between flanking copies of mini-Mu. R-prime plasmids could be transferred easily into different recipients and were used in complementation studies. PstI restriction digests of four R-prime plasmids carrying P. fluorescens 6.2 DNA showed a number of additional bands, suggesting that several genes were transposed together with the selected marker on the plasmid.

Alcaligenes↗