Genetic transfer of lithoautotrophy mediated by a plasmid-cointegrate from Pseudomonas facilis

Pseudomonas facilis (DSM 620) is host of two plasmids one of which (pHG22-a) has been shown to be involved in lithoautotrophic metabolism. The lithoautotrophic marker was transferred via conjugation to mutants of two wild type strains of P. facilis and to the heterotrophic bacterium Pseudomonas delafieldii. The transfer required mobilization by the IncP1 plasmid RP4. Transconjugants contained a plasmid which neither correlated in size with RP4 nor with pHG22-a. This newly formed plasmid, pHG22-c, was shown to be a cointegrate consisting of RP4 DNA and a 50-kb insert derived from the native plasmid pHG22-a. DNA-DNA hybridization using lithoautotrophic genes of Alcaligenes eutrophus as DNA probes, revealed the presence of hydrogenase structural and regulatory genes in addition to genes of autotrophic carbon dioxide fixation on the cointegrate pHG22-c.     

Conjugal transfer of hydrogen-oxidizing ability of Alcaligenes hydrogenophilus to Pseudomonas oxalaticus

Conjugal transfer of hydrogen-oxidizing ability (Hox) of the hydrogen bacterium Alcaligenes hydrogenophilus was examined. Intraspecific cross of plasmid pHG21-a that encodes hydrogenases that mediate hydrogen oxidation was most frequent at 25 C; the optimal temperature for growth was 30 C. The plasmid could be transferred from A. hydrogenophilus to Pseudomonas oxalaticus OX1 and OX4, and the resulting strains gained the capacity for autotrophic growth with H2 and CO2. Plasmid pHG21-a was maintained in P. oxalaticus OX1 and OX4 as stably as in A. hydrogenophilus.     

Hydrogen autotrophy of Nocardia opaca strains is encoded by linear megaplasmids

Several linear megaplasmids were detected in the facultatively lithoautotrophic Gram-positive bacterium Nocardia opaca. The wild-type strain MR11 contains, in addition to the cccDNA plasmids pHG31-a and pHG31-b, the linear plasmids pHG201 (270 kb), pHG202 (400 kb) and pHG203 (420 kb). The wild-type strain MR22 contains, in addition to the cccDNA plasmid pHG33, the linear plasmids pHG204 (180 kb), pHG205 (280 kb) and pHG206 (510 kb). After preparation of DNA from cells embedded in agarose, the linear plasmids were demonstrated by pulsed-field electrophoresis. By means of DNA probes for genes of soluble hydrogenase and ribulose-bisphosphate carboxylase, the conjugative plasmids pHG201 and pHG205 were shown to be the carriers of the genetic information for these enzymes. A restriction map of pHG201 for the enzymes AsnI, SpeI, XbaI is presented.     

No correlation exists between the conjugative transfer of the autotrophic character and that of plasmids in Nocardia opaca strains

A new isolate of Nocardia opaca was obtained by enrichment culture for aerobic lithoautotrophic growth on CO2 and H2. This strain, MR22, is very similar to N. opaca MR11 (formerly 1b) in functioning as a donor for genetic information determining the ability to grow lithoautotrophically (Aut character) in matings with Aut- strains of N. opaca or closely related heterotrophic species. The strain contains a plasmid, pHG33 of about 110 kb. A mutant was isolated from strain MR22 which was plasmid-free, and had lost the Aut character, resistance to 50 microM-thallium salt and susceptibility to the nocardia-specific bacteriophage phi B1. As a recipient of the Aut character, this plasmid-free mutant was as well suited as plasmid-bearing Aut- strains of N. opaca. In matings with the mutant as recipient the frequency of Aut+ transconjugants per donor was 3 X 10(-4) with N. opaca MR11 (pHG31-a, Aut+, Tlr, Strs, phi B1s) and 2 X 10(-3) with N. opaca MR22 (pHG33, Aut+, Tlr, Strs, phi B1r) as donor. Phenotypic characterization of the transconjugants, which had been selected for the Aut marker, revealed that in many cases the Aut marker had been transferred without plasmid transfer. Furthermore, plasmid-free, Aut+ transconjugants functioned as donors for the Aut marker. Both plasmid-free and plasmid-bearing transconjugants transferred the Aut marker to the Aut- strains of N. opaca with a frequency which was one or two orders of magnitude higher than that of the wild-type strains. The plasmids pHG31-a and pHG33 code for thallium resistance (50 microM-thallium acetate). The frequency of thallium-resistant transconjugants was 10(-1) to 10(-2) per donor; all thallium-resistant transconjugants contained the donor plasmid. We conclude that the plasmids pHG31-a of strain MR11 and pHG33 of strain MR22 of N. opaca carry the genetic information for thallium resistance but not the Aut character. As plasmid-free Aut+ strains can function as donors the Aut character is assumed to reside on the chromosome and to function as an independent self-transmissible genetic element.     

Isolation of hydrogenase regulatory mutants of hydrogen-oxidizing bacteria by a colony-screening method

Mutants derepressible for hydrogenases (Hox d) have been isolated from the wild type of Alcaligenes hydrogenophilus which is inducible for hydrogenases (Hox i). The mutants are able to form the hydrogenases during growth on gluconate under air while the wild type requires molecular hydrogen for hydrogenase systhesis.Mutant selection involved alternating growth under autotrophic and heterotrophic conditions. Mutants derepressed for hydrogenases after growth on gluconate were recognized by a new colony-screening method allowing differentiation between colonies of hydrogenase-containing and hydrogenase-free cells of aerobic hydrogen-oxidizing bacteria. The method is based on the ability of the colonies to reduce triphenyltetrazolium chloride in the presence of monoiodoacetate and gaseous hydrogen to its water-insoluble purple formazan. Endogenous dye reduction (under nitrogen) and the function of the cytoplasmic NAD-reducing hydrogenase were completely inhibited by monoiodoacetate. The applicability of the method has been demonstrated for wild type strains and mutants of various hydrogen-oxidizing bacteria. When mutants of A. hydrogenophilus and A. eutrophus H16 lacking the Hox-encoding plasmids pHG21-a and pHG1, respectively, were used as recipients and Hox d mutant M 201 of A. hydrogenophilus as a donor transconjugants appeared which had received the Hox d character and the megaplasmid pHG21-a.Abbreviations MIAc monoiodoacetate - TTC 2,3,5-triphenyl-2-tetrazolium chloride - Hox ability to oxidize hydrogen Dedicated to Gerhard Drews on the occasion of his 60th birthday, remembering the education and inspiration we received from our teacher Johannes Buder at the Martin-Luther University of Halle     

Identification of new peptides synthesized under the hydrogenase control system of Alcaligenes eutrophus

Total protein of Alcaligenes eutrophus was analyzed by two-dimensional protein map. Cells grown at 30° C expressed hydrogen-oxidizing (Hox) ability mediated by a soluble (Hos) and a particulate hydrogenase (Hop). Hox ability was not expressed at 37° C (HoxTs). The six subunits of the two hydrogenases were identified. Besides these six subunits eight peptides were not or hardly detected at 37° C. The mutant HF117 which expressed Hox ability at 37° C (HoxTr), formed the hydrogenase peptides and five of the eight peptides. These peptides designated B, C, E, F, and H were characterized by their isoelectric point and molecular mass (M _r); their M _r were 18 800, 45 400, 41 900, 39 400, and 40 600, respectively. The five peptides were not formed in regulatory Hox^– mutants, and not formed in mutants cured of plasmid pHG1, carrying the genetic information for hydrogenase formation. Strain HF160, carrying a Tn5 insertion in a gene essential for Hos expression specifically did not form the B-peptide. All peptides were found in the soluble fraction of cell extracts, the F-peptide was also detected in the particulate fraction. The function of the new Hox-peptides is presently unknown.Abbreviations PAGE polyacrylamide gelelectrophoresis - SDS sodium dodecylsulfate - Hox hydrogen oxidizing ability     

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 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.     

In vivo generation of R68.45-pPGH1 hybrid plasmids conferring a Phl+ (meta pathway) phenotype

Summary Plasmid pPGH1 originating from Pseudomonas putida strain H carries all the genes required for the degradation of phenol (or cresols) via the meta cleavage pathway. Besides mobilization of pPGH1 by a plasmid of the incompatibility group P-1, hybrid plasmids conferring the Phl⁺ phenotype could be selected, when R68.45 was the conjugative plasmid. The hybrids contain the complete R68.45 and part of pPGH1. Integration of Phl-DNA of pPGH1 into R68.45 occurred exclusively via the IS21 region of R68.45.Dedicated to Udo Taubeneck on the occasion of his 60th birthday     

Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli

Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS21, at a frequency of 10^?3–10^?5 per donor cell. Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21. No resolution of cointegrates of pACYC184 and the P-type plasmid could be found in recA recipient cells. In the cointegrate, the E. coli plasmid is flanked by single copies of IS21 in direct orientation. After resolution of the cointegrate in recA⁺ recipients, the mobilizing plasmid R68.45 lost one copy of IS21 becoming indistinguishable from plasmid R68. It was shown that during mobilization, insertion element IS21 transposes to the mobilized plasmid. Insertion sites and orientations of IS21 in 33 pACYC184::IS21 insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYC184 in different regions but only in one orientation. The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H₂ consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H₂ evolution and the light-dependent H₂ production in the presence of thiosulfate. Both Hox⁺ and Hup⁺ mutants demonstrated light-dependent H₂ uptake stimulated by CO₂ but only the Hup⁺ mutant was able to mediate O₂-dependent H₂ consumption in the dark. The ability of the Hox⁺ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO₂, H₂, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Transfer and expression of the herbicide-degrading plasmid pJP4 in aerobic autotrophic bacteria

Plasmid pJP4 encoding the ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (Tfd⁺) was transferred by conjugation from Escherichia coli JMP397 to various lithoautotrophic strains of Alcaligenes eutrophus and to the autotrophic bacterium Pseudomonas oxalaticus. The herbicide-degrading function of the plasmid was phenotypically expressed in all of the recipients. The majority of Tfd⁺ transconjugants also exhibited additional plasmid-encoded properties such as 3-chlorobenzoate degradation, resistance to mercuric ions, and sensitivity to the male-specific bacteriophage PR11. Furthermore, Tfd⁺ transconjugants were able to act as donors of plasmid pJP4. Physical evidence is presented by agarose gel electrophoresis showing that plasmid pJP4 coexisted with the resident plasmids widely distributed in this group of bacteria. However, in some of the hosts plasmid pJP4 was not stably maintained, had a reduced size and tended to form multimers.     

Genetic analysis of the parB+ locus of plasmid R1

Summary Megaplasmid DNA from mutants has been analysed physically for deletions and insertions in order to identify the location of hydrogenase (hox) genes in Alcaligenes eutrophus. Four classes of mutants have been examined: mutants defective in genes coding for soluble NAD-dependent hydrogenase (hoxS), mutants impaired in the membrane-bound hydrogenase (hoxP), mutants altered in the regulation of hox gene expression (hoxC) and mutants with lesions in the carbon dioxide fixing enzyme system (cfx). A comparison of the restriction patterns with EcoRI, BamHI and HindIII, complementation studies with cloned DNA and DNA - DNA hybridization experiments showed that genes coding for hox and cfx are clustered on a 100-kb region of the 450-kb plasmid pHG1.     

Involvement of megaplasmids in heterotrophic derepression of the carbon-dioxide assimilating enzyme system in Alcaligenes spp.

The facultatively chemolithoautotrophic hydrogen-oxidizing bacteria Alcaligenes eutrophus and Alcaligenes hydrogenophilus partially derepressed the formation of phosphoribulokinase and ribulosebisphosphate carboxylase during heterotrophic growth on fructose or gluconate. We examined whether the indigenous magaplasmids in these bacteria that encode the ability to oxidize hydrogen affected this derepression. The results suggest an involvement of the plasmids in the derepression for the following reasons: (i) wild-type strains, except A. eutrophus TF93, exhibited the derepressible phenotype; (ii) plasmid-cured mutants formed the enzymes with formate as autotrophic growth substrate but did not derepress their formation during heterotrophic growth; (iii) the phenotype of the wild type was restored by transfer of the plasmids into plasmid-cured mutants. Plasmid pHG2 from strain TF93 differed from the other wild-type plasmids by conferring a non-derepressible phenotype onto the harboring strain. Mutants of A. eutrophus H16 carrying deletions in plasmid pHG1 showed a similar phenotype as that of the plasmid-cured mutants. We concluded that the plasmids from the various strains studied encode a regulatory ability to derepress phosphoribulokinase and ribulosebisphosphate carboxylase under heterotrophic growth conditions.Abbreviations PRK phosphoribulokinase - RuBPC ribulosebisphosphate carboxylase - Hox ability to oxidize hydrogen - Cfx ability to fix carbon dioxide autotrophically Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

The insertion sequence IS21 of R68.45 and the molecular basis for mobilization of the bacterial chromosome

The IncP-1 plasmid mutant R68.45, which is able to mobilize the chromosomes of many Gram-negative bacteria, was shown to carry a 2.10-kb insertion sequence designated IS21. This sequence transposed to the small multicopy plasmid pED815 at a high frequency (2 × 10^?3) and in two pED815::IS21 derivatives inactivated the tetracycline-resistance and replication functions, respectively. We propose that the chromosome-mobilizing ability of R68.45 is due to the formation of an R68.45-chromosome cointegrate during transposition of IS21. This would account for its high efficiency and the absence of a fixed chromosomal origin of transfer in Pseudomonas aeruginosa PAO, and its ability to function in a variety of bacterial hosts. R68.45 is formed from R68 by duplication of a 2.1-kb DNA segment including a distinctive cluster of seven restriction endonuclease sites. The two copies of the duplicated segment are probably contiguous and so might have arisen by a transition type of mechanism. IS21 is similar in length to the duplicated segment and includes the same set of seven cleavage sites located at similar distances from the two termini. However, the single copy of the duplicated segment in R68 transposed at an undetectably low frequency (<6 × 10^?8); either the duplicated segment and IS21, although overlapping, are not identical, or they are identical but the transposition system is nonfunctional in R68. Our further investigations of R68.45 and of several independently isolated chromosome-mobilizing derivatives of R68 demonstrated that these were indistinguishable from each other and that they did not include any P. aeruginosa PAO DNA. Furthermore, we searched without success for sequences corresponding to IS21, and to the Escherichia coli K-12 insertion sequences IS1, IS2, and IS3, on the chromosomes of P. aeruginosa PAO and PAT and P. putida PPN, and on several Pseudomonas plasmids. The contribution of homology to low-frequency chromosomal mobilization by these plasmids is discussed.     

Determinants encoding resistance to several heavy metals in newly isolated copper-resistant bacteria

Three copper-resistant, gram-negative bacteria were isolated and characterized. Of the three strains, Alcaligenes denitrificans AH tolerated the highest copper concentration (MIC = 4 mM CuSO(4)). All three strains showed various levels of resistance to other metal ions. A. denitrificans AH contains sequences which cross-hybridized with the mer (mercury resistance) determinant of Tn21 and the czc (cobalt, zinc, and cadmium resistance), cnr (cobalt and nickel resistance), and chr (chromate resistance) determinants of A. eutrophus CH34. DNA-DNA hybridization with probes prepared from A. eutrophus CH34 and Tn21 revealed the presence of chr-, cnr-, and mer-like sequences on the 200-kb plasmid pHG27 and of czc, cnr, and mer homologs located on the chromosome. The second strain, classified as Alcaligenes sp. strain PW, carries czc, cnr, and mer homologs on the 240-kb plasmid pHG29-c and a chr determinant on the 290-kb plasmid pHG29-a; a third plasmid, the 260-kb large plasmid pHG29-b, is cryptic. In contrast to the Alcaligenes strains, which were isolated from metal-contaminated water, Pseudomonas paucimobilis CD was isolated from the air. This strain harbors two cryptic plasmids: the 210-kb large plasmid pHG28-a and the 40-kb plasmid pHG28-b. Southern analysis revealed no homology between the metal ion resistance determinants of A. eutrophus CH34 and P. paucimobilis CD.     

The effect of sulfur compounds on H<Subscript>2</Subscript> evolution/consumption reactions,mediated by various hydrogenases,in the purple sulfur bacterium, <Emphasis Type="Italic">Thiocapsa roseopersicina</Emphasis>

The influence of reduced sulfur compounds (including stored S⁰) on H₂ evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S₂O₃²⁻, SO₃²⁻, S²⁻ and S⁰ as electron donors for light-dependent H₂ production. Dark H₂ evolution from organic substrates via Hox hydrogenase was inhibited by S⁰. Under light conditions, endogenous H₂ uptake by Hox or Hup hydrogenases was suppressed by S compounds. СО₂-dependent H₂ uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process. Dark H₂ consumption via Hyn hydrogenase was connected to utilization of S⁰ as an electron acceptor and resulted in the accumulation of H₂S. In wild type BBS, with high levels of stored S⁰, dark H₂ production from organic substrates was significantly lower, but H₂S accumulation significantly higher, than in the mutant GB1121(Hox⁺). There is a possibility that H₂ produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S⁰.     

Transfer of IncP Plasmids to Extremely Acidophilic Thiobacillus thiooxidans

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and and pUB307 were transferred directly to extremely acidophilic Thiobacillus thiooxidans from Escherichia coli by conjugation at frequencies of 10^-5 to 10^-7 per recipient. The ability of T. thiooxidans to receive and express the antibiotic resistance markers was examined. The plasmid RP4 was transferred back to E. coli from T. thiooxidans at a frequency of 1.0 × 10^-3 per recipient.     

Heterotrophic bacteria of the freshwater neuston and their ability to act as plasmid recipients under nutrient deprived conditions

Significantly higher numbers of Gram-negative heterotrophic bacteria were present at the air-water interface (neston) of freshwater lakes than in the bulk water. Neuston bacteria were distinguished as a population distinct from bacteria in the bulk water by a higher incidence of pigmented colony types and significantly greater levels of multiple resistance to antibiotics and heavy metals. The incidence of plasmids in 236 neuston and 229 bulk water strains were similar (14 and 16.2%, respectively). Nine of 168 plasmid-free strains and 2 of 14 plasmid carrying strains, isolated from both bulk water and neuston, acted as recipients of plasmid R68.45 in plate matings with aPseudomonas aeruginosa donor strain PAO4032 at 21°C, but at frequencies below that of matings with a restriction-minus recipient strain ofP. aeruginosa, strain PAO1168. In a model system composed of nutrient-free synthetic lake water, plasmid R68.45 was shown to transfer betweenP. aeruginosa strains at frequencies between 10⁻³ and 10⁻⁵. Transconjugants were detected about 100 times more frequently at the interface than in the bulk water, which in part reflected a greater enrichment of the donor at this site. None of the aquatic isolates were able to act as recipients of plasmid R68.45 in this model system with strain PAO4032 as donor. The results suggest that under nutrient deprived conditions, the spread of plasmid R68.45 and similar plasmids by lateral transfer into this particular aquatic population would be a rare event.