- Volume 134, Issue 4, 1988

Whole organisms of Thiobacillus tepidarius oxidize thiosulphate to sulphate with the obligatory formation of tetrathionate as an intermediate. Oxidation of thiosulphate to tetrathionate shows an apparent K m of about 120 m and is relatively insensitive to FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), HQNO (2-heptyl-4-hydroxyquinoline-N-oxide) or thiocyanate. Oxidation of tetrathionate to sulphate shows a K m of about 27 m and is strongly inhibited by FCCP, HQNO, thiocyanate and gramicidin. Sulphite oxidation is also inhibited by FCCP and HQNO. Trithionate oxidation to sulphate occurred and showed unexplained dependence on the presence of sulphate ions. A H+/O quotient of about 4 for proton translocation driven by substrate oxidation was seen for each of thiosulphate, tetrathionate and sulphite. ATP synthesis coupled to thiosulphate oxidation was completely abolished by FCCP. The results obtained are consistent with the oxidation of thiosulphate (and probably trithionate) to tetrathionate in the periplasm of the cell, with HQNO-insensitive electron transport to cytochrome c, and with further oxidation of tetrathionate (and sulphite) to sulphate after FCCP-sensitive transport to the cytoplasmic side of the membrane. The latter oxidations involve HQNO-sensitive electron transport via cytochrome b. Inhibition of tetrathionate metabolism by thiocyanate and gramicidin would be consistent with tetrathionate transport by a S4O-/4H+ symport process. The proton translocation experiments indicate the mechanism of H+ extrusion to depend on electron transfer within the quinone/cytochromes bc segment of the respiratory chain, and does not involve a proton-pumping oxidase. The sulphur-compound-oxidizing system of T. tepidarius is shown to be quite different from that previously described for T. versutus.

The enzyme oxidizing thiosulphate to tetrathionate (‘tetrathionate synthase’) has been purified: the native enzyme has a M r of 138000 and subunit M r of 45000, contains no haem and shows K m values for thiosulphate of 4 and 110 μm respectively with cytochrome c or ferricyanide as electron acceptor. Most of the enzyme was recovered from the periplasm of the cell. Evidence is also presented for a trithionate hydrolyase, catalysing the hydrolytic cleavage of trithionate to thiosulphate and sulphate. This activity was partially purified and assayed in a coupled system in which cytochrome c reduction by the purified tetrathionate synthase was measured. Tetrathionate oxidation by cell-free preparations could not be obtained, but a sulphite dehydrogenase (with ferricyanide as electron acceptor) was demonstrated and sulphite-dependent reduction of cytochrome c in T. tepidarius membrane preparations occurred. The latter was inhibited by HQNO (2-heptyl-4-hydroxyquinoline-N-oxide), indicating the involvement of cytochrome b. Sulphite oxidation by APS reductase (adenylylsulphate reductase) could not be detected, which was consistent with the earlier demonstration of complete inhibition of ATP synthesis in intact organisms by FCCP (carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone). A scheme is presented which is consistent with all the whole organism and enzyme data available to date.

In previous work it had been shown that Acinetobacter calcoaceticus wild-type strain NCIB 8250 had only an l-mandelate deydrogenase but it could give rise to mutants that contained an evolved d-mandelate dehydrogenase; conversely, wild-type strain EBF 65/65 had only a d-mandelate dehydrogenase but gave rise to mutants that possessed an evolved l-mandelate dehydrogenase. Several other wild-type strains of A. calcoaceticus have now been shown to grow on both enantiomers of mandelate. In every case the l-mandelate dehydrogenases were found to be much more heat-stable and insensitive to inhibition by p-chloromercuribenzoate than were the d-mandelate dehydrogenases when measured in bacterial extracts. All the d-mandelate dehydrogenases in the wild-type strains were inactivated to about the same extent by an antiserum that had been raised in a rabbit against an evolved d-mandelate dehydrogenase. An evolved d-mandelate deydrogenase (from a mutant strain derived from strain NCIB 8250) and an original d-mandelate dehydrogenase (from a mutant strain derived from strain EBF 65/65) were purified to homogeneity by the same procedure and were indistinguishable as judged by immunological cross-reactivity of the native and the sodium-dodecyl-sulphate-denatured enzymes, solubility in cholate, net charge at pH 7·5, pI value, salting-out properties, M r value, apparent K m value for d-mandelate, heat-stability and sensitivity to p-chloromercuribenzoate. The most likely explanation for the appearance of evolved mandelate dehydrogenases in strains of A. calcoaceticus is that cryptic genes become expressed.

The developmental cycle of the obligate intracellular prokaryote Chlamydia trachomatis involves the serial alternation of two distinct morphological forms of the organism. To examine the basis of chlamydial differentiation we have searched for developmentally regulated gene products in this species. Chlamydia-infected cells were pulse-labelled with [35S]cysteine at various stages of development and the products of synthesis examined by SDS-PAGE. Our results indicate that the synthesis of the cysteine-rich outer-membrane proteins is developmentally regulated, occurring only late in the cycle during the conversion of reticulate bodies to elementary bodies. Both hydroxyurea and ampicillin block this conversion; as a result of this blockade the cysteine-rich outer-membrane proteins are not produced in the presence of either drug.

Auxotrophic mutants of Rhizobium meliloti 104A14 were isolated using nitrous acid mutagenesis followed by penicillin enrichment. Mutants in ornithine transcarbamylase, argininosuccinate synthetase or serine-glycine biosynthesis formed nitrogen-fixing (Fix+) nodules on the roots of alfalfa (Medicago sativa). Mutants with defects in ornithine, pyrimidine, purine, asparagine, leucine, methionine or tyrosine biosynthesis, in one-carbon metabolism or in carbamoylphosphate synthetase formed nodules but these nodules were unable to fix nitrogen. Prototrophic revertants were always Fix+. Plasmids that would complement many of these auxotrophs were isolated by transduction with a P2 cosmid gene bank of R. meliloti 104A14. These plasmids were then introduced into mutants of the same and different classes and the growth and symbiotic phenotypes of the new strains were determined. In all cases, complementation of the nutritional defect restored symbiotic nitrogen fixation.

We have previously isolated ineffective (Fix-) mutants of Rhizobium meliloti 104A14 requiring both arginine and uracil, and thus probably defective in carbamoylphosphate synthetase. We describe here the molecular and genetic analysis of the R. meliloti genes coding for carbamoylphosphate synthetase. Plasmids that complement the mutations were isolated from a R. meliloti gene bank. Restriction analysis of these plasmids indicated that complementation involved two unlinked regions of the R. meliloti chromosome, car A and car B. Genetic complementation between the plasmids and mutants demonstrated a single complementation group for car A, but two overlapping complementation groups for car B. The cloned R. meliloti genes hybridize to the corresponding E. coli car A and car B genes which encode the two subunits of carbamoylphosphate synthetase. Transposon Tn5 mutagenesis was used to localize the car A and car B genes on the cloned R. meliloti DNA. The cloned R. meliloti car A and car B genes were unable to complement E. coli car A or car B mutants alone or in combination. We speculate on the mechanism of the unusual pattern of genetic complementation at the R. meliloti car B locus.

The results presented extend previous investigations on the genetics of nitrogen fixation in Azotobacter chroococcum and indicate that nif- and fix-like DNA is located in at least five different regions of the genome. Region I contains functional copies of nifS, V and M, as well as nifH, D and K, all of which complemented mutants of Klebsiella pneumoniae. In addition, nifE-and/or nifN-like and nifU-like DNA is located in this region. The organization of the nif cluster in region I closely resembles that of K. pneumoniae, though spread over 22 kb as compared with 14 kb. Region II contains a functional nifB gene, which complemented a K. pneumoniae nifB mutant, and seems to be adjacent to a nifA-like gene. Region III harbours nifH *, encoding a second nitrogenase Fe-protein. Region IV contains a reiteration of nifE- and/or nifN-like sequences, and DNA homologous to Rhizobium meliloti fix ABC is present in region V. The apparent complexity of nif DNA in A. chroococcum is probably related to the two systems for N2-fixation present in this organism.

The dichloromethane (DCM)-utilizing facultative methylotroph Methylobacterium sp. DM4 was shown to contain three plasmids with approximate sizes of 120 kb, 40 kb and 8 kb. Curing experiments suggested that the DCM-utilization character was correlated with the possession of an intact 120 kb plasmid. The DCM-utilization genes were cloned on the broad-host-range vector pVK100. Plasmid pME1510, a recombinant plasmid carrying a 21 kb HindIII fragment complemented DCM-utilization-negative derivatives of Methylobacterium sp. DM4 and conferred the DCM-utilization-positive phenotype to a number of Gram-negative methylotrophic bacteria. In Southern hybridization experiments with pME1510 as a probe, chromosomal DNA from Methylobacterium sp. DM4 gave definite signals while purified plasmid DNA did not. Plasmid pME1510 did not hybridize with total DNA from a cured DCM-non-utilizing derivative of Methylobacterium sp. DM4. It is concluded that the DCM-utilization genes are located on the chromosome or on a megaplasmid. Curing procedures thus led to the formation of a chromosomal or megaplasmid deletion larger than 21 kb and covering the DCM-utilization genes or to the loss of an undetected megaplasmid.

Cured derivatives of Salmonella dublin and S. typhimurium showed reduced virulence following oral infection of mice (104–105-fold for S. dublin, 102-fold for S. typhimurium). Large plasmids from S. dublin and S. typhimurium independently restored virulence to the cured S. dublin but truncated S. dublin plasmids with deletions in a previously identified virulence region did not. This common virulence region identified in plasmids from S. dublin and S. typhimurium was shown to be carried on plasmids from 11 other serotypes of Salmonella but was absent from 10 plasmid-containing serotypes. TnA and Tn10 were transduced from the virulence region of two TnA-insertion mutants of S. dublin and one Tn10-insertion mutant of S. typhimurium that showed diminished virulence to recipient wild-type strains of S. dublin, S. enteritidis and S. typhimurium. Each transductant showed a decrease in mouse virulence within the range 103–105. It is therefore proposed that similar virulence determinants are expressed in different serotypes. It was also shown that integration that occurred during curing was Tn10 dependent.

The role of lipopolysaccharide (LPS) in the susceptibility of Escherichia coli and Salmonella typhimurium to non-immune human serum was investigated using serum-sensitive strains of both enterobacteria. LPS from serum-resistant strains of E. coli and S. typhimurium could activate and completely remove the serum bactericidal activity, and also showed dose-dependent anti-complement activity. These properties were mainly due to the high-molecular-mass LPS: the low-molecular-mass LPS from serum-resistant strains of E. coli and S. typhimurium had only a slight effect on the serum bactericidal activity, and showed only low anti-complement activity, even at high concentration. The results suggest that LPS composition, especially the O-antigen polysaccharide chains, contributes to the susceptibility of E. coli and S. typhimurium strains to complement-mediated serum bactericidal activity.

Three varieties of Xenorhabdus nematophilus subsp. nematophilus released lipopolysaccharide (LPS) during bacteraemia in larval Galleria mellonella. Larval serum triggered release of LPS from the bacterial envelope. LPS activated the plasmatocytes and eventually damaged the haemocytes. LPS and its lipid A portion bound to the insect haemocytes through d-glucosamine-binding lectins on the haemocyte surfaces. The toxicity of LPS resides in the fatty acids of the lipid A moiety.

Melanogenesis in Cryptococcus neoformans begins with the oxidation of dihydroxyphenylalanine by the enzyme phenol oxidase. The succeeding steps are very rapid. Two intermediates, dopachrome and 5,6-dihydroxyindole, have been isolated and characterized by high performance liquid chromatography. A pathway of melanin formation in C. neoformans is proposed, based on the presence of these intermediates.

Pseudomonas saccharophila, grown microaerobically in batch culture, showed much higher uptake hydrogenase activity in N2-fixing than in NH+ 4-grown cultures. Hydrogenase synthesis was induced by H2 under a low partial pressure of O2 and under air, in autotrophic as well as in heterotrophic conditions, provided that the sucrose concentration was relatively low. Sucrose at 15 mm repressed hydrogenase formation but did not inhibit preformed activity. Other utilizable carbon substrates repressed, while non-utilizable substrates did not repress, hydrogenase synthesis. The activity, unlike nitrogenase, was not sensitive to O2 but hydrogenase synthesis was partially repressed by O2.

d-Arginine dehydrogenase activity was discovered in Pseudomonas aeruginosa. This enzyme was inducible by its substrate, d-arginine, as well as by its product, 2-ketoarginine, but not by l-arginine. The enzyme activity was measured in vitro, in the presence of artificial electron acceptors (phenazine methosulphate and iodonitrotetrazolium chloride). 2-Ketoarginine was catabolized further to 4-guanidinobutyraldehyde, 4-guanidinobutyrate and 4-aminobutyrate. Two enzymes involved, 4-guanidinobutyraldehyde dehydrogenase and guanidinobutyrase, were inducible by 2-ketoarginine; the latter enzyme was also strongly induced by 4-guanidinobutyrate. An arginine racemase activity was detected by an in vivo test. d-Arginine had the potential to be catabolized via the d-arginine dehydrogenase pathway and, after racemization, via the three l-arginine catabolic pathways previously demonstrated in P. aeruginosa. In mutants blocked in the l-arginine succinyltransferase pathway, but not in the wild-type, l-arginine was channelled partially into the d-arginine dehydrogenase pathway. Mutations in the kauB locus abolished growth of P. aeruginosa on 2-ketoarginine, agmatine and putrescine, and led to loss of 4-guanidinobutyraldehyde dehydrogenase and 4-aminobutyraldehyde dehydrogenase activities. Thus, these two activities appear to be due to one enzyme in P. aeruginosa. The kauB locus was mapped on the chromosome between lysA and argB and was not linked to known genes involved in the three l-arginine catabolic pathways. The existence of four arginine catabolic pathways illustrates the metabolic versatility of P. aeruginosa.