Sequence analysis of the chromosomal and plasmid genes encoding phosphoribulokinase from Alcaligenes eutrophus

Two DNA fragments encoding the chromosomal and plasmid copies of the gene (cfxP) encoding phosphoribulokinase (PRK) from the chemoautotrophic bacterium Alcaligenes eutrophus, were sequenced and found to be highly homologous. The gene (cfxF) of another Calvin cycle enzyme, fructose-1,6-bisphosphatase (FBPase), was identified as terminating immediately upstream of cfxP, but was not completely contained on both fragments. A hypothetical, also incompletely contained, open reading frame starts closely downstream from cfxP. Genes cfxF, cfxP, and the third hypothetical gene seem to belong to the same operon. The cfxP genes encode highly homologous PRK isoenzyme subunits consisting of 292 aa residues with calculated Mrs of 33 319 (chromosomal PRKc) and 33 164 (plasmid-encoded PRKp). There is little overall sequence similarity between the bacterial and plant (spinach) PRK, apart from some structural motifs.     

Denitrification by Alcaligenes eutrophus is plasmid dependent.

Curing of the hydrogenase-specifying megaplasmid pHG indigenous to strains of the facultative lithoautotrophic bacterium Alcaligenes eutrophus was correlated with a loss of denitrifying ability (Nitd). The retransfer of plasmid pHG1 reconstituted the Nitd phenotype. Plasmid-free mutants were still capable of converting some nitrate to nitrite, but they did not metabolize nitrite under anaerobic conditions.     

Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus.

Resistances to chromate and cobalt were cloned on a 30-kilobase-pair (kb) DNA region from the large Alcaligenes eutrophus plasmid pMOL28 into the broad-host-range mobilizable cosmid vector pVK102. A restriction nuclease map of the 30-kb region was generated. The resistances expressed from the hybrid plasmids after transfer back into A. eutrophus were inducible and conferred the same degree of resistance as the parent plasmid pMOL28. Resistances were expressed in metal-sensitive Alcaligenes strains and related bacteria but not in Escherichia coli. Resistance to chromate was further localized on a 2.6-kb EcoRI fragment, and resistance to cobalt was localized on an adjoining 8.5-kb PstI-EcoRI fragment. When the 2.6-kb EcoRI fragment was expressed in E. coli under the control of a bacteriophage T7 promoter, three polypeptides with molecular masses of 31,500, 21,000, and 14,500 daltons were visible on autoradiograms. The 31,500- and 21,000-dalton polypeptides were membrane bound; the 14,500-dalton polypeptide was soluble.     

Control of autotrophic carbon assimilation in Alcaligenes eutrophus by inactivation and reactivation of phosphoribulokinase

Phosphoribulokinase in Alcaligenes eutrophus was partially inactivated when an autotrophic culture was shifted to heterotrophic growth with pyruvate as the sole source of carbon and energy. A similar response was observed on addition of various organic substrates to autotrophic cultures during the transition to mixotrophic growth. The extent of inactivation depended on the added substrate. Pyruvate or lactate caused the strongest inactivation among the tested substrates. Up to 75% of the phosphoribulokinase activity found in the autotrophic cells was lost within 30 min after supplementation of the cultures with either of these two substrates. This loss of enzyme activity was not the result of degradation of enzyme protein. Inactivation of phosphoribulokinase was accompanied by a decrease in the CO2 fixation rate of the cells. Reactivation of the enzyme occurred after exhaustion of pyruvate from the medium. Neither inactivation nor reactivation required de novo protein synthesis; however, continued energy conversion was necessary for the inactivation to occur. We suggest that the pyruvate metabolism of A. eutrophus is involved in these regulatory processes which act on phosphoribulokinase. They appear to contribute to the control of autotrophic CO2 assimilation in this organism.     

Alcaligenes eutrophus hydrogenase genes (Hox)

Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.     

Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34.

A 238-kilobase-pair plasmid, pMOL30, confers resistance to cadmium, zinc, and cobalt salts in Alcaligenes eutrophus CH34. After Tn5 mutagenesis, restriction nuclease analysis, and Southern DNA-DNA hybridization, a 9.1-kilobase-pair EcoRI fragment was found to harbor all of these resistance properties and was cloned into the broad-host-range hybrid plasmid pRK290. When transferred to a plasmid-free derivative of CH34, the hybrid plasmid conferred the same degree of resistance as the parent plasmid pMOL30. In two other Alcaligenes strains, the hybrid plasmid was expressed, but to a lower degree than in CH34 derivatives.     

Chromosomal locations of three Bacillus subtilis din genes.

Previously isolated DNA damage-inducible (din) genes of Bacillus subtilis have been mapped on the bacterial chromosome by bacteriophage PBS1-mediated transduction. The din genes have been localized to three positions on the B. subtilis map. dinA cotransduction with the hisA locus was 80%, while dinC cotransduction with this marker was about 56%. dinB is unlinked to hisA, but its cotransduction with the dal-1 and purB loci was 84 and 22%, respectively.     

Nickel transport in Alcaligenes eutrophus

Transport of nickel ions was studied in Alcaligenes eutrophus. Two transport systems for nickel ions exist to satisfy the nickel demand for the lithotrophic hydrogen metabolism. A major nickel transport activity exhibited an apparent affinity constant (K _m) of 17 M nickel chloride. This activity was competitively inhibited by Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺. A minor nickel transport activity was determined in the presence of high (0.8 mM) magnesium. This activity was not inhibited by Zn²⁺ or Mn²⁺; its K _m was determined to be 0.34 M nickel chloride. These kinetics suggested a second transport system in A. eutrophus. The membrane potential of A. eutrophus was decreased upon the addition of ammonium ions leading to a decreased nickel transport. This inhibition could be reversed by fructose or by hydrogen indicating an energy dependent nickel transport. Protonophores inhibited the nickel transport. However, inhibitors of ATP synthase like dicyclohexylcabodimide or venturicidin had little or no effect on nickel transport. These data indicated that the transport was coupled to the proton motive force.     

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.     

Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients.

This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes.     

Respiratory components and oxidase activities in Alcaligenes eutrophus.

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H2 oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H2 oxidase by 50% and solubilizes the bulk of the ATPase. 2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa3, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm). 3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2--3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H2 oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20-100% stimulation of oxygen uptake). 4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.     

Glycollate production and excretion by Alcaligenes eutrophus.

Autotrophic cultures of the facultative chemolithotroph Alcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO2 to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction of D-ribulose 1,5-diphosphate carboxylase under the same conditions.     

Quantitative requirements for exponential growth of Alcaligenes eutrophus.

Quantitative nutrient requirements for unrestricted autotrophic growth of Alcaligenes eutrophus were determined. Minimum saturating concentrations of Mg2+, SO42-, PO43-, Fe3+, and Na2+ for an optical density increase of 2 were 10(-4) M 8 X10(-5) M, 5 X 10(-4) to 6 X 10(-4) M, 10(-5) M, and 10(-7) to 2 X 10(-7) M, respectively. Trace metal requirements for cobalt, chromium, and copper were also demonstrated, but minimum concentrations could not be determined because other reagents contributed a high background of these metals. Under certain conditions an apparent response to zinc was observed, although other experiments suggest the zinc salt contained another metal that was required for growth. Poly-beta-hydroxybutyrate biosynthesis was shown to be initiated by a magnesium or sulfate deficiency as well as by a nitrogen or phosphate deficiency.     

Dense autotrophic cultures of Alcaligenes eutrophus.

Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter.     

Dense autotrophic cultures of Alcaligenes eutrophus.

Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter.     

An oxygen-binding flavohemoprotein from Alcaligenes eutrophus.

A procedure is described for the purification of a soluble flavohemoprotein from the hydrogen bacterium Alcaligenes eutrophus. The isolated protein exists as a monomer with a molecular weight of approx. 43,000. The molecule contains two prosthetic groups, 1 mol each of noncovalently bound FAD and protoheme per monomer. The absorption spectra of the protein in its ferric, ferrous-deoxy and ferrous-carboxy forms are similar to those of hemoglobins, with the exception of the flavin contribution (absorption maxima--ferric form: 395, 456, 483, 645 nm; ferrous-deoxy form: 436, 560 nm; ferrour-CO form: 423, 539, 569 nm). The flavohemoprotein when reduced by NADH in aerobic solution is capable of binding oxygen reversibly. The stable oxygenated complex exhibits absorption maxima at 414, 541, and 576 nm. The protein catalyzes the reduction of various dyes and cytochrome c by NADH.     

Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.