Structural gene (nirS) for the cytochrome cd1 nitrite reductase of Alcaligenes eutrophus H16.

Denitrification by Alcaligenes eutrophus H16 is genetically linked to megaplasmid pHG1. Unexpectedly, the gene encoding the nitrite reductase (nirS) was identified on chromosomal DNA. The nirS product showed extensive homology with periplasmic nitrite reductases of the heme cd1-type. Disruption of nirS abolished nitrite-reducing ability, indicating that NirS is the enzyme essential for denitrification in A.eutrophus.     

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.     

Tn5-induced mutations affecting sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha

Mutants of Thiosphaera pantotropha defective in chemolithoautotrophic growth were obtained by transpositional mutagenesis with Tn5 coding for kanamycin resistance. The suicide vehicle for introducing Tn5 to T. pantotropha was pSUP5011 harbored by Escherichia coli. Kanamycin-resistant isolates were screened for the inability to grow with reduced sulfur compounds (Sox-). Four classes of Sox- mutants were obtained. Three were of different pleiotropic phenotypes: (i) unable to grow with formate, nitrate, and xanthine; (this class strongly suggested the involvement of a molybdenum cofactor in inorganic sulfur-oxidizing ability); (ii) no growth with hydrogen; (iii) slight growth with hydrogen and formate. Two plasmids, pHG41 (about 450 kilobase pairs) and pHG42 (110 kilobases), were identified in lysates of T. pantotropha. In one Sox- mutant pHG41 could not be detected. Revertant analysis suggested that pHG41 and pHG42 were not involved in the Sox character.     

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.     

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.     

A physical map of the megaplasmid pHG1, one of three genomic replicons in Ralstonia eutropha H16

We have used pulsed field gel electrophoresis and megabase DNA techniques to investigate the basic genomic organization of Ralstonia eutropha H16, and to construct a physical map of its indigenous megaplasmid pHG1. This Gram-negative, soil-dwelling bacterium is a facultative chemolithoautotroph and a denitrifier. In the absence of organic substrates it can grow on H2 as its sole energy source and CO2 as its sole source of carbon. Under anaerobic conditions it can utilize nitrate as a terminal electron acceptor, whereby dinitrogen is released. Essential genetic determinants of the enzyme systems responsible for these metabolic processes are linked to the 0.44-Mb conjugative megaplasmid pHG1. Aside from pHG1, the genome of R. eutropha H16 is comprised of two circular chromosomes measuring 4.1 and 2.9 Mb, adding up to a total genome size of 7.1 Mb. An estimated five copies of rDNA are distributed on the two chromosomes. A macrorestriction map of pHG1 was derived for the endonucleases DraI and XbaI. Hybridization studies showed that genes for anaerobic metabolism are located on all three genomic replicons.     

Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor

The B subunit of verotoxin (VT1B) from enterohemorrhagic Escherichia coli is responsible for the attachment of the holotoxin to the cell surface, by binding to the glycolipid, globotriaosyl ceramide. After receptor-mediated endocytosis, the toxin is targeted to the Golgi complex by a process of retrograde transport. We took advantage of this unique property of VT1B to measure the pH of the Golgi complex in intact live cells. Purified recombinant VT1B was labeled with either rhodamine or fluorescein for subcellular localization by confocal microscopy. After 1 h at 37 degrees C, VT1B accumulated in a juxtanuclear structure that colocalized with several Golgi markers, including alpha-mannosidase II, beta-COP, and NBD-ceramide. Moreover, colchicine and brefeldin A induced dispersal of the juxtanuclear staining, consistent with accumulation of VT1B in the Golgi complex. Imaging of the emission of fluorescein-labeled VT1B was used to measure intra-Golgi pH (pHG), which was calibrated in situ with ionophores. In intact Vero cells, pHG averaged 6.45 +/- 0.03 (standard error). The acidity of the Golgi lumen dissipated rapidly upon addition of bafilomycin A1, a blocker of vacuolar-type ATPases, pHG remained constant despite acidification of the cytosol by reversal of the plasmalemmal Na+/H+ antiport. Similarly, pHG was unaffected by acute changes in cytosolic calcium. Furthermore, pHG recovered quickly toward the basal level after departures imposed with weak bases. These findings suggest that pHG is actively regulated, despite the presence of a sizable H+ "leak" pathway. The ability of VT1B to target the Golgi complex should facilitate not only studies of acid-base regulation, but also analysis of other ionic species.     

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.     

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.     

Electron microscopic localization of hydrogenase genes on the megaplasmid pHG 1 in Alcaligenes eutrophus

Summary Plasmids carrying hydrogenase genes in Alcaligenes eutrophus wild type H 16 and in two transposon Tn5 —induced mutants have been investigated by electron microscopy. Besides the pHG1 megaplasmid (458±27 kb) carrying genes coding for structural and regulatory properties of hydrogenases, small plasmids of unknown significance have been detected. The sizes of EcoRI fragments obtained from pHG1 were measured from electron micrographs. They were significantly different from sizes determined previously by agarose gel electrophoresis.Plasmid pHG1 isolated from the wild type H 16 was shown to contain two inverted repeats (IR 16-1 and IR 16-2) with sizes similar to known transposons.From electron microscopic hybridization studies, it was deduced that the sites of insertion of Tn5 into a regulation gene on pHG1 for both soluble and membrane-bound hydrogenase, and of Tn5-Mob into the gene coding for structural properties of the soluble hydrogenase, are about 67.2 kb apart. One of the inverted repeats (IR 16-1) was localized in between these sites.     

In vivo cloning of genes determining lithoautotrophy (Aut) on a plasmid from Alcaligenes hydrogenophilus

Hydrogenase (hox) genes on the megaplasmid pHG21-a from Alcaligenes hydrogenophilus, whose lithoautotrophic growth (Aut) is supported by H₂-oxidation (Hox) and CO₂-fixation (Cfx), were cloned in vivo using a broad host range IncP1 plasmid R68.45. The recombinant plasmid was detected by the characteristic that it was transferred at a frequency 10⁶-fold higher than pHG21-a in intrastrain mating of the Hox⁻ Cfx⁺ bacterium Pseudomonas oxalaticus OX1. All of six recombinant plasmids designated pFUs inherited all three resistance markers of R68.45. Four plasmids (pFU3, pFU8, pFU11, and pFU15) with a molecular size of 69 Md had only membrane-bound hydrogenase (hoxP) genes, and two plasmids (pFU7 and pFU9) of 85 Md had both hoxP and soluble hydrogenase (hoxS) genes. The Hox⁻ Cfx⁻ bacteria P. oxalaticus OX4 and OX6 gained Aut⁻ phenotype by the possession of pHG21-a, pFU7 or pFU15. These results showed that Hox plasmid pHG21-a was an Aut plasmid and pFU7 and pFU15 inherited this phenotype, pFU7 was maintained stably in P. oxalaticus OX1 and had all of the lithoautotrophic phenotypes of pHG21-a. pFU7, rather than pHG21-a, is useful for further studies on the transfer of the Aut phenotype to a broad range of bacteria.     

In vivo plasmid construction by integrative recombination within a 156 bp sequence homology

pHG165, a pBR322 copy number derivative of pUC8, has a 38-bp polylinker multiple cloning site located at the 5′ end of lacα. A further 156 bp, 3′ to the multiple cloning site, completes the coding sequence for the production of the β galactosidase α peptide. We describe the use of in vivo plasmid-chromosone cointegrates as a construction method that in this instance has enabled us to cross out the pHG165 multiple cloning site to obtain a wild-type β-galactosidase sequence for the α peptide. Because the DNA sequence available for homologous recombination was only 156 bp in length, the frequency of crosses that removed the multiple cloning site was less than 1 × 10⁻⁹. These crosses were easily obtained, however, after amplification by ampicillin selection.     

Cloning and expression in Escherichia coli of a recA-like gene from Bacteroides fragilis

The recombinant plasmid pHG100, containing a 5.2-kb DNA fragment from Bacteroides fragilis, complemented defects in homologous recombination, DNA repair and prophage induction to various levels in an Escherichia coli recA mutant strain. There was no DNA homology between the cloned B. fragilis recA-like gene and E. coli chromosomal DNA. pHG100 produced two proteins with Mr of approx. 39,000 and 37,000 which cross-reacted with antibodies raised against E. coli RecA protein. The production of these proteins was not increased after UV induction. The cloned B. fragilis recA-like gene product did not enhance the production of native but defective E. coli RecA protein after UV irradiation.     

Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H(2)-based ithoautotrophy and anaerobiosis

The self-transmissible megaplasmid pHG1 carries essential genetic information for the facultatively lithoautotrophic and facultatively anaerobic lifestyles of its host, the Gram-negative soil bacterium Ralstonia eutropha H16. We have determined the complete nucleotide sequence of pHG1. This megaplasmid is 452,156 bp in size and carries 429 potential genes. Groups of functionally related genes form loose clusters flanked by mobile elements. The largest functional group consists of lithoautotrophy-related genes. These include a set of 41 genes for the biosynthesis of the three previously identified hydrogenases and of a fourth, novel hydrogenase. Another large cluster carries the genetic information for denitrification. In addition to a dissimilatory nitrate reductase, both specific and global regulators were identified. Also located in the denitrification region is a set of genes for cytochrome c biosynthesis. Determinants for several enzymes involved in the mineralization of aromatic compounds were found. The genes for conjugative plasmid transfer predict that R.eutropha forms two types of pili. One of them is related to the type IV pili of pathogenic enterobacteria. pHG1 also carries an extensive "junkyard" region encompassing 17 remnants of mobile elements and 22 partial or intact genes for phage-type integrase. Among the mobile elements is a novel member of the IS5 family, in which the transposase gene is interrupted by a group II intron.     

Transformation of Rhodosporidium toruloides

Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae.     

Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.     

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     

Comparative characterization of repABC-type replicons of Paracoccus pantotrophus composite plasmids

The repABC replicons have an unusual structure, since they carry genes coding for partitioning (repA, repB) and replication (repC) proteins, which are organized in an operon. So far, the presence of these compact bi-functional modules has been reported only in the megaplasmids of the Rhizobiaceae and within the plasmid pTAV1 (107kb) of Paracoccus versutus. We studied the distribution of repABC-type replicons within bacteria belonging to the genus Paracoccus. We found that repABC replicons occur only in the group of pTAV1-like plasmids: pKLW1, pHG16-a, pWKS2, and pPAN1, harbored by different strains of Paracoccus pantotrophus. A partial sequencing approach followed by phylogenetic analysis revealed that these replicons constitute a distinct evolutionary branch of repABC replicons. Incompatibility studies showed that they represent two incompatibility groups designated IncABC1 (pTAV1, pKLW1, and pHG16-a) and IncABC2 (pPAN1). Sequence comparison using available databases allowed the identification, within plasmid pRS241d of Rhodobacter sphaeroides 2.4.1, of an additional sequence highly homologous to the paracoccal repABC replicons, which has been included in comparative analyses.     

Transfer and Expression of Lithoautotrophy and Denitrification in a Host Lacking These Metabolic Activities

The conjugative 450-kilobase-pair megaplasmid pHG1 from Alcaligenes eutrophus H16 was transferred to the herbicide-degrading soil bacterium A. eutrophus JMP134. This transfer was achieved by means of RP4 mobilization and a Tn5-Mob insertion provided in trans on the megaplasmid replicon. Although kanamycin-resistant transconjugants also occurred with other gram-negative species such as Rhizobium, Agrobacterium, and thiobacteria, A. eutrophus JMP134 was the only recipient which stably maintained the megaplasmid. pHG1-containing transconjugants derived from JMP134 expressed all metabolic functions associated with the plasmid: the ability to oxidize hydrogen through catalysis of two hydrogenases, to assimilate carbon dioxide via the Calvin cycle pathway, and to grow with nitrate anaerobically. All of these metabolic activities were absent in the original strain JMP134.