Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha.

For the identification of the DNA region responsible for the sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha, we used previously isolated Tn5-mob insertional Sox- mutants. For seven mutants, the Tn5-mob insertion was localized on the chromosome rather than on the megaplasmids pHG41 or pHG42 by using the Tn5-mob-harboring vehicle pSUP5011 as probe. The specific insertion of Tn5-mob into a sox gene was determined for one Sox- mutant, strain TP19. An 18-kb EcoRI fragment was cloned in Escherichia coli by using the mobilizable plasmid pSUP202 as vector and the kanamycin resistance gene of Tn5 as marker. Conjugal transfer of the resulting hybrid plasmid, pKS3-13, to the wild type resulted in two phenotypically different groups of recombinants. Ninety-five percent of the recombinants were Sox+, kanamycin resistant, and tetracycline resistant; 5% were homogenote recombinants exhibiting the Sox-, kanamycin-resistant, tetracycline-sensitive phenotype, and these indicated the specific insertion. To isolate the respective wild-type sox gene, total DNA from a heterogenote recombinant was partially restricted with EcoRI, religated, and transformed in E. coli. Transformants carrying a pSUP202-derived hybrid plasmid with the intact sox gene were identified by screening for a tetracycline-resistant, kanamycin-sensitive, and chloramphenicol-sensitive phenotype and by complementation of the Sox- mutant TP19. A plasmid of this type, pEG12, contained an insert of 13 kb which gave a positive signal in Southern hybridization with the homologous probe of pKS3-13. pEG12 was used to determine the DNA homology of the sulfur-oxidizing enzyme systems of other thiobacteria. Strong hybridization signals were obtained with total DNA of the neutrophilic sulfur-oxidizing bacteria Paracoccus denitrificans, Thiobacillus versutus, and Rhodobacter capsulatus. No hybridization signal was obtained with DNA of other neutrophilic or acidophilic thiobacteria examined.     

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.     

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.     

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.     

The effect of electron acceptor variations on the behaviour of Thiosphaera pantotropha and Paracoccus denitrificans in pure and mixed cultures

Abstract The competitive advantages provided by a capacity for aerobic denitrification have been tested by comparing Thiosphaera pantotropha (which denitrifies aerobically and anaerobically), with a strain of Paracoccus denitrificans (which only denitrifies under anaerobic conditions) in acetate-limited chemostats. A comparison of μ -C_s curves based on K _s and μ _max measurements indicated that Pa. denitrificans could be expected to dominate mixtures of the two species at high growth rates when the dissolved oxygen was above 80% of air saturation and NH₃ was the sole source of nitrogen. The comparison also suggested t that at lower growth rates, lower dissolved oxygen tensions, and/or in the presence of nitrate, Tsa. pantotropha should have the competitive advantage. Chemostat experiments with mixtures of the two species showed that Tsa. pantotropha did, indeed, dominate the population when expected. However, when Pa. denitrificans was expected to dominate, only a small increase in the Pa. denitrificans numbers was possible before Tsa. pantotropha formed a biofilm on the walls of the chemostat instead of washing out, and was again able to out-compete Pa. denitrificans for acetate. Experiments with axenic chemostat cultures subjected to aerobic/anaerobic switches showed that Tsa. pantotropha , with its constitutive denitrifying system, was able to adjust smoothly to the changing environmental conditions and thus continued to grow. Pa. denitrificans does not have constitutive denitrifying enzymes, and could consequently not adjust its metabolism to the lack of oxygen rapidly enough. It therefore washed out at a rate equivalent to the dilution rate.     

Isolation and Sequence Analysis of the Sox-1, -2, -3 Homologs in Trionyx sinensis and Alligator sinensis Having Temperature-Dependent Sex Determination

Members of the Sox gene family are characterized by an HMG-box that shows sequence similarity with that of the mouse testis-determining gene Sry. Using degenerate primers PCR, seven and eight HMG-box motifs of Sry-related genes were cloned and sequenced from genomic DNA of Trionyx sinensis (termed TS41-47) and Alligator sinensis (AS41-48) with TSD (temperature-dependent sex determination). Among 15 Sry-related genes, TS41, TS42, AS41, and AS42 shared 80, 72, 81, and 79% amino acid identity, respectively, with each HMG-box domain of the mouse Sox-1, -2, and -3 genes by Blast analysis. Molecular phylogenetic analysis showed that the clustering of TS41-42 and AS41-42 was distant to the clustering of the nonreptilian vertebrate Sox-1, -2, -3 homologs, including fish, amphibian, bird, and mammals. The amino acid identity among TS41-42, AS41-42, and the nonreptilian vertebrate Sox-1, -2, -3 homologs is lower than identities among the Sox-1, -2, -3 homologs, suggesting that the sequence changes in TS41-42 of Trionyx sinensis and AS41-42 of Alligator sinensis might have occurred after the diversification of amniotes.     

Isolation and sequence analysis of the Sox-1, -2, -3 homologs in Trionyx sinensis and Alligator sinensis having temperature-dependent sex determination

Members of the Sox gene family are characterized by an HMG-box that shows sequence similarity with that of the mouse testis-determining gene Sry. Using degenerate primers PCR, seven and eight HMG-box motifs of Sry-related genes were cloned and sequenced from genomic DNA of Trionyx sinensis (termed TS41-47) and Alligator sinensis (AS41-48) with TSD (temperature-dependent sex determination). Among 15 Sry-related genes, TS41, TS42, AS41, and AS42 shared 80, 72, 81, and 79% amino acid identity, respectively, with each HMG-box domain of the mouse Sox-1, -2, and -3 genes by Blast analysis. Molecular phylogenetic analysis showed that the clustering of TS41-42 and AS41-42 was distant to the clustering of the nonreptilian vertebrate Sox-1, -2, -3 homologs, including fish, amphibian, bird, and mammals. The amino acid identity among TS41-42, AS41-42, and the nonreptilian vertebrate Sox-1, -2, -3 homologs is lower than identities among the Sox-1, -2, -3 homologs, suggesting that the sequence changes in TS41-42 of Trionyx sinensis and AS41-42 of Alligator sinensis might have occurred after the diversification of amniotes.     

Methanol and methylamine utilization result from mutational events in Thiosphaera pantotropha

With choline as carbon source Thiosphaera pantotropha GB17 grew with a doubling time (t_d) of 6 h. The cellular yield was 55.8 g dry cell weight per mol of choline, indicating that its methyl moieties were used for growth. However, T. pantotropha was unable to grow with methanol or with methylamine as carbon source. Mutants were isolated from liquid or from solid media able to grow with methanol (Mox⁺) as carbon or methylamine as nitrogen source (Mam⁺). The Mox⁺ mutant GB17M grew with a mean t_d of 11.7h and a growth yield of 8.9 g dry cell weight per mol of methanol. Diauxic growth of strain GB17M was observed with mixtures of pyruvate and methanol as substrates in batch culture. Methanol led to the formation of methanol dehydrogenase, formate dehydrogenase, ribulosebisphosphate carboxylase and of a soluble cytochrome c-551.5. Tn5-insertional mutants defective in the thiosulfate oxidizing enzyme system or in hydrogenase acquired the Mox⁺ phenotype. However, Tn5-insertional mutants defective in either a c-type cytochrome or the molybdenum cofactor did not mutate to the Mox⁺ phenotype, indicating common functions in thiosulfate and in methanol metabolism.     

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     

Growth of Thiobacillus ferrooxidans on Formic Acid

A variety of acidophilic microorganisms were shown to be capable of oxidizing formate. These included Thiobacillus ferrooxidans ATCC 21834, which, however, could not grow on formate in normal batch cultures. However, the organism could be grown on formate when the substrate supply was growth limiting, e.g., in formate-limited chemostat cultures. The cell densities achieved by the use of the latter cultivation method were higher than cell densities reported for growth of T. ferrooxidans on ferrous iron or reduced sulfur compounds. Inhibition of formate oxidation by cell suspensions, but not cell extracts, of formate-grown T. ferrooxidans occurred at formate concentrations above 100 μM. This observation explains the inability of the organism to grow on formate in batch cultures. Cells grown in formate-limited chemostat cultures retained the ability to oxidize ferrous iron at high rates. Ribulose 1,5-bisphosphate carboxylase activities in cell extracts indicated that T. ferrooxidans employs the Calvin cycle for carbon assimilation during growth on formate. Oxidation of formate by cell extracts was NAD(P) independent.     

Syntrophic growth on formate: a new microbial niche in anoxic environments

Anaerobic syntrophic associations of fermentative bacteria and methanogenic archaea operate at the thermodynamic limits of life. The interspecies transfer of electrons from formate or hydrogen as a substrate for the methanogens is key. Contrary requirements of syntrophs and methanogens for growth-sustaining product and substrate concentrations keep the formate and hydrogen concentrations low and within a narrow range. Since formate is a direct substrate for methanogens, a niche for microorganisms that grow by the conversion of formate to hydrogen plus bicarbonate--or vice versa--may seem unlikely. Here we report experimental evidence for growth on formate by syntrophic communities of (i) Moorella sp. strain AMP in coculture with a thermophilic hydrogen-consuming Methanothermobacter species and of (ii) Desulfovibrio sp. strain G11 in coculture with a mesophilic hydrogen consumer, Methanobrevibacter arboriphilus AZ. In pure culture, neither Moorella sp. strain AMP, nor Desulfovibrio sp. strain G11, nor the methanogens grow on formate alone. These results imply the existence of a previously unrecognized microbial niche in anoxic environments.     

Involvement of host DNA gyrase in growth of bacteriophage T5.

Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB [gyrB(Ts)], but it did grow in gyrA(Ts) mutants at 42 degrees C. These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5. The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase. However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit. This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host. The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K. N. Kreuzer and N. R. Cozzarelli, J. Bacteriol. 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C. We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5. Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication. In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated.     

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.     

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.     

Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

We report the complete genome of Thermofilum pendens, a deeply branching, hyperthermophilic member of the order Thermoproteales in the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact, T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features that are common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known previously to utilize peptides as an energy source, but the genome revealed a substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may obtain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogen lyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time that this enzyme has been found outside the Methanosarcinales, and the presence of a presenilin-related protein. The predicted highly expressed proteins do not include proteins encoded by housekeeping genes and instead include ABC transporters for carbohydrates and peptides and clustered regularly interspaced short palindromic repeat-associated proteins.     

1,3‐Propanediol production from glycerol by a newly isolated Trichococcus strain

A coccal bacterium (strain ES5) was isolated from methanogenic bioreactor sludge with glycerol as the sole energy and carbon source. Strain ES5 fermented glycerol to 1,3‐propanediol as main product, and lactate, acetate and formate as minor products. The strain was phylogenetically closely related to Trichococcus flocculiformis; the rRNA gene sequence similarity was 99%. However, strain ES5 does not show the typical growth in chains of T. flocculiformis. Moreover, T. flocculiformis does not ferment glycerol. Strain ES5 used a variety of sugars for growth. With these substrates, lactate, acetate and formate were the main products, while 1,3‐propanediol was not formed. The optimum growth temperature of strain ES5 ranges from 30–37°C, but like several other Trichoccoccus strains, strain ES5 is able to grow at low temperature (< 10°C). Therefore, strain ES5 may be an appropriate catalyst for the biotechnological production of 1,3‐propanediol from glycerol at low ambient temperature.