Characterization of the cell wall and outer membrane of Rhodopseudomonas capsulata.

Sucrose density gradient centrifugation of cell envelopes of chemotrophically grown cells of Rhodopseudomonas capsulata St. Louis (= ATCC 23782) resulted in the separation of a cytoplasmic membrane from a cell wall fraction (buoyant densities, 1.139 and 1.215 g/cm3, respectively). The cell wall fractions (untreated or Triton extracted) contained peptidoglycan- and lipopolysaccharide-specific components. Their neutral sugar content, mainly rhamnose and galactose, was high (250 and 100 micrograms/mg [dry weight] of material) due to a non-lipopolysaccharide polymer. The fatty acid content was low (less than or equal to 60 micrograms/mg [dry weight] of material), and half of it was contributed by lipopolysaccharide (3-OH-C10:0, C12:1, and 3-oxo-C14:0). The predominant other fatty acid was C18:1. An outer membrane fraction, obtained by lysozyme treatment of the Triton-extracted cell wall, showed essentially the same chemical composition except for almost complete removal of peptidoglycan. Saline extraction (0.9% NaCl, 37 degrees C, 2 h) removed a lipopolysaccharide-protein(-phospholipid?) complex from whole cells of R. capsulata St. Louis. The polypeptide patterns of the cell wall and outer membrane as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comprised 20 to 25 different polypeptides (most of them very faint) and were dominated by a single, heat-modifiable major protein (Mr 69,000 after solubilization below 60 degrees C; Mr 33,000 at temperatures above 70 degrees C).     

Chemical cross-linking studies of the light-harvesting pigment-protein complex B800-850 of Rhodopseudomonas capsulata

The spatial relationship of the three polypeptides contained in the B800-850 light-harvesting complex of Rhodopseudomonas capsulata has been studied with chemical cross-linking of crude membrane preparations of the phototrophic negative mutant strain Y5. Samples were cross-linked with the cleavable reagent dithiobis (succinimidyl propionate) (1.1 nm chain length) and analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Membranes labelled with 14C-amino acids were used to determine the compositional stoichiometry of cross-linked products. It was found that the two polypeptides with an apparent Mr of 8000 and 10 000, respectively, that are associated with the pigments bacteriochlorophyll a and carotenoid form homooligomers as well as heterooligomers. The data support the idea that these polypeptides are closely arranged in clusters probably containing at least four of each species. The third subunit with an Mr of 14 000, which is not associated with pigments, was found to be most susceptible to cross-linking and formed homooligomers but no heterooligomers with the other two subunits, and is thus likely to be loosely attached to these clusters. Comparative studies with the phototrophic positive wild type strain indicated that the results found with the phototrophic negative mutant strain Y5 reflect the organization of the B800-850 complex in the membrane of Rhodopseudomonas capsulata. Studies with the isolated B800-850 complex revealed that the sterical arrangement of the three constituent polypeptides in dodecyl dimethylamine-N-oxide containing solutions must be very similar to that in the membrane.     

Different degradation pathways for glucose and fructose in Rhodopseudomonas capsulata

In Rhodopseudomonas capsulata the enzymes of the Entner-Doudoroff pathway and the Embden-Meyerhof pathway have been examined. Fructose-grown cells contained inducible activities of phosphoenolpyruvate-fructosephospho-transferase and 1-phosphofructokinase and only low levels of fructokinase and 6-phosphofructokinase. Although fructose-grown cells contained, in addition, all the enzymes of the Entner-Doudoroff pathway together with fructose-1,6-diphosphatase and phosphoglucose isomerase, the Entner-Doudoroff pathway was not operative in fructose catabolism and served only the degradation of glucose. The functional separation of glucose and fructose catabolism via the Entner-Doudoroff and a modified Embden-Meyerhof pathway, respectively, was confirmed by different approaches: 1. Radiorespirometric experiments with glucose and fructose labelled in positions 1, 2, 3, 3+4 and 6 have been carried out. The pattern of 14CO2-evolution from position-labelled glucose was characteristic for the Entner-Doudoroff pathway, that from position-labelled fructose for the Embden-Meyerhof pathway. 2. In the presence of arsenite up to 50% of glucose- and fructose-carbon was excreted as pyruvate. Using 1-14C-glucose, 86% of the pyruvate was labelled in the carboxyl group, whereas using 1-14C-fructose only 19% of the pyruvate was labelled in the carboxyl group. 3. A glucose-6-phosphate dehydrogenase-deficient mutant was isolated which lacked a functional Entner-Doudoroff pathway but which was unaltered in its ability to grow on fructose.     

Oxidative degradation of purines by the faculative phototrophic bacterium Rhodopseudomonas capsulata

The mechanism of purine degradation was studied in the facultative phototrophic bacterium Rhodopseudomonas capsulata. Using tungstate as an inhibitor of synthesis of an active xanthine dehydrogenase it could be shown in growth experiments that purine compounds are transformed to uric acid as central purine intermediate prior to ring cleavage. Because of its rapid degradation, the mechanism of uric acid conversion was investigated using 1-methyluric acid as substrate. The analogue was partially degraded by whole cells yielding 3-methylallantoin and methylurea. This implicated an oxidative degradation of 1-methyluric acid analogous to oxidation of uric acid to allantoin suggesting uric acid degradation via allantoin. In cell-free extracts, allantoinase, allantoicase, ureidoglycolase and urease activities degrading allantoin to NH₃, CO₂ and glyoxylic acid were detected. Apparently, purine degradation in R. capsulata proceeds in a manner similar to many aerobic microorganisms. It is peculiar to this bacterium, however, that the pathway evidently operates also under anaerobic conditions. In cell extracts, oxidation of uric acid was observed which could be increased by addition of cytochrome c. The basis of this stimulation is still unknown.     

Characterization of Rhodopseudomonas capsulata

Thirty-three strains of Rhodopseudomonas capsulata have been studied in order to develop a more comprehensive characterization of the species. On the basis of morphological, nutritional, physiological and other properties, the characteristics of an ideal biotype have been defined, which can be used to distinguish Rps. capsulata from similar purple bacteria. In this connection, two properties of Rps. capsulata are of particular note: a) sensitivity to penicillin G is 10³–10⁵ times greater than that shown by closely related species, and b) all strains examined are susceptible to lysis by one or more strains of host species-specific virulent bacteriophages. It appears that members of the species Rps. capsulata form a stringent taxonomic grouping.     

Porin isolated from the cell envelope of Rhodopseudomonas capsulata.

The isolate major outer membrane protein from Rhodopseudomonas capsulata St. Louis (ATCC 23782) has a high porin activity in reconstituted phospholipid liposomes. The pore size of the homooligomeric porin with subunits of Mr 33,000 was determined to be about 0.8 nm in radius. Circular dichroism data revealed major portions of the beta structure. Heating of the oligomer resulted in monomer formation, loss of porin activity (60 to 70 degrees C), and change to alpha structure (100 degrees C).     

Urea: An intermediate of aerobic and anaerobic purine degradation in Rhodopseudomonas capsulata

Abstract A mutant strain ( pur ^-) defective in utilization of purines was isolated from Rhodopseudomonas capsulata . In the mutant, the loss of purine utilization correlated with urease deficiency. In contrast to the wild-type strain, the mutant catalyzed release of urea from purines. The nitrogen of the purine ring was completely liberated as urea indicating that the latter compound is an intermediate of the purine degradation pathway in Rps. capsulata . The degradation pattern was identical under aerobic and anaerobic conditions.     

Glycerol-utilizing mutants of Rhodopseudomonas capsulata

The isolation and study of glycerol-utilizing mutants of Rhodopseudomonas capsulata indicated that the wild-type organism has genes capable of coding for the catabolism of glycerol but is unable to express them. Furthermore, the genetic lesion in the original glycerol-utilizing mutant, L1, occurred very close to these genes.     

Mutational analysis of serine-glycine biosynthesis in Rhodopseudomonas capsulata.

Rhodopseudomonas capsulata possesses the enzymes of both the "phosphorylated" and the "non-phosphorylated" pathways of serine biosynthesis. Certain mutants with lesions in the phosphorylated pathway are serine-glycine auxotrophs, though they still produce enzymes of the non-phosphorylated sequence. These results indicate that the phosphorylated pathway is essential for the synthesis of serine and glycine in R. capsulata under the condtions tested.     

DNA-DNA hybridization of Rhodopseudomonas capsulata,Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably a R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.Abbreviations GC guanosine + cytosine - SSC standard saline citrate buffer     

Respiratory deficient mutants of Rhodopseudomonas capsulata

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c into the medium and lack cytochrome c ₂.Abbreviations Ap ampicillin - CIE crossed immunoelectrophoresis - cyt cytochrome - Cm chloramphenicol - Km kanamycin - SDS sodium dodecylsulfate - Tc tetracycline     

Potassium transport system of Rhodopseudomonas capsulata

Rhodopseudomonas capsulata required potassium (or rubidium or cesium as analogs of potassium) for growth. These cations were actively accumulated by the cells by a process following Michaelis-Menten saturation kinetics. The monovalent cation transport system had Km's of 0.2 mM K+, 0.5 mM Rb+, and 2.6 mM Cs+. The rates of uptake of substrates by the potassium transport system varied with the age of the culture, although the affinity constant for the substrates remained constant. The maximal velocity of uptake of K+ was lower in aerobically grown cells than in photosynthetically grown cells, although the Km's for K+ and for Rb+ were about the same.     

Mapping of Rhodopseudomonas capsulata nif genes

The endogenous gene transfer system of Rhodopseudomonas capsulata was used to analyze mutations which block the ability to use molecular nitrogen as the sole nitrogen source (nif). With this fine-structure mapping tool, linkage of nif mutations could be reliably established if separated by 2,700 base pairs or less. Eleven independent mutations were analyzed, and five linkage groups were found. The overall chromosomal arrangement of these groups awaits conjugational or physical analysis. A candidate for the inactive subunit of R. capsulata Fe protein was located in gels at a position of about 38,000 molecular weight, 5,000 more than that of the presumed active subunit.     

-Aminolevulinic acid dehydratase of Rhodopseudomonas capsulata

δ-Aminolevulinic acid dehydratase, an enzyme which catalyzes the synthesis of the pyrrole, porphobilinogen, from two molecules of δ-aminolevulinic acid, has been purified 400-fold from Rhodopseudomonas capsulata. Some of its properties were compared to the enzyme from Rhodopseudomonas spheroides and to those from eucaryotic cells. The enzyme of R. capsulata appears to be both similar to that of R. spheroides in some respects and also similar to those of eucaryotic cells. The enzyme from R. capsulata does not require metallic cations for activation, is not inhibited by EDTA, and is insensitive to inhibition by hemin and protoporphyrin.     

Characterization of the plasmid DNAs of Rhodopseudomonas capsulata

Presence of extrachromosomal DNa in Rhodopseudomonas capsulata strain BH9 was shown by the appearance of a satellite band in a dye-buoyant density gradient. Radioactively labelled DNA was prepared from this satellite band and examined on a 5–20% sucrose gradient. Three radioactive peaks with sedimentation coefficients of 100 S, 94 S, and 58–64 S, respectively, were consistently observed. Analysis of these sedimentation coefficients suggested that there are two species of plasmid DNA with molecular sizes of 94×10⁶ daltons (named pBH91) and 74×10⁶ daltons (named pBH92). The 58–64 S peak is attributed to open circular molecules. DNAs from each peak of the sucrose gradient were examined by electronmicroscopy, and the results agree closely with those of the sucrose gradient analysis. Reassociation kinetics of the plasmid DNA was also followed. Addition of total DNA of strain BH9 increased the renaturation rate of the plasmid DNA. It was calculated from the magnitude of the increase that approximately 10% of the BH9 total DNA may hybridize with the plasmid sequences. DNA prepared from the gene transfer agent (GTA) produced by R. capsulata increases the renaturation rate of the plasmid to the same extent as total DNA isolated from the GTA producing strain, Y262.     

Chemical and ultrastructural studies on the cell wall ofStaphylococcus simulans biovarstaphylolyticus

Cell walls of strains ofStaphylococcus simulans biovarstaphylolyticus andS. aureus FDA 209P were compared ultrastructurally and chemically to investigate the mechanism of resistance of this strain ofS. simulans to its own staphylolytic endopeptidase. Chemical analysis of the peptidoglycans of the various strains examined showed that cells that were more resistant to lysis by the endopeptidase had lower glycine/serine ratios in their cross bridges and that, within a species, the more resistant cells had either fewer residues in these cross bridges or fewer cross bridges. Ultrastructural studies showed that cell wall thickness was not involved in resistance to the enzyme. Comparisons of the endopeptidase susceptibility of intact cells and isolated peptidoglycans from these cells suggested that the three-dimensional structure of the cell wall may play a role in resistance to lysis by the endopeptidase.     

Enzymes of serine biosynthesis in Rhodopseudomonas capsulata

Rhodopseudomonas capsulata has been shown to possess all the enzymatic activities of both the phosphorylated and nonphosphorylated pathways of serine biosynthesis. In addition there was an active serine hydroxymethyltransferase which catalyzed the reversible interconversion of serine and glycine. In cells grown photosynthetically with malate as the carbon source, the activities of the phosphorylated pathway enzymes were substantially higher than the analogous reactions of the nonphosphorylated sequence. l-Serine (1 mm) caused approximately 60%, inhibition of the first enzyme of the phosphorylated route, 3-phosphoglyceric acid dehydrogenase, but was less effective in inhibiting the last enzyme, phosphoserine phosphatase. Glycine also exerted a regulatory effect on this pathway but it was not as potent an inhibitor as serine. The inhibitions caused by serine and glycine were simply additive; there was no evidence of concerted feedback inhibition of the phosphorylated pathway by these amino acids.