Regulation of catalase synthesis in Proteus mirabilis]

During the log-phase growth of Proteus mirabilis the specific activity of catalase decreases, while at the beginning of or during the stationary phase an increase takes place which is abolished by inhibitors of nucleic acid or protein synthesis. Glucose in the culture medium has no appreciable effect on the level of enzyme synthesis nor does the passage of bacteria to anaerobiosis bring any noticeable change. Successive additions of hydrogen peroxide up to weak final concentrations (0.2--0.5 mM) stimulate catalase synthesis. Determination of the enzyme in vivo reveals but a weak proportion of the total catalase which can only be titrated after the breakdown of cells. The titrable enzyme in vivo represents, as an order of magnitude, the activity found associated with the cell wall, in an easily released form after the mechanical separation of the inner and outer membranes. Thus, bacteria can act upon exogenous peroxide only through a peripheral catalase while they possess in a masked form an important reserve of cytoplasmic enzyme.     

Regulation of reductase formation in Proteus mirabilis

Summary The levels of several redox enzymes in a chlorate-resistant mutant of Proteus mirabilis, which is partially affected in the formation of formate hydrogenlyase, thiosulfate reductase and tetrathionate reductase, were compared with those of the wild type. The composition of the electron transport system of both strains was almost the same in cells grown aerobically, but very different in cells grown anaerobically. In the mutant, the cytochrome content increased twofold, whereas the level of the anaerobic enzymes is strongly diminished. The anaerobic formation of electron transport components in the mutant was, in contrast to that of the wild type, not influenced significantly by azide. During anaerobic growth with nitrate low levels of a functional nitrate reductase system were formed in the mutant. Under these conditions the formation of formate dehydrogenase, formate hydrogenlyase, formate oxidase, thiosulfate reductase, tetrathionate reductase, cytochrome b_563,5 and partly that of cytochrome a₂, was repressed. The repressive effect of nitrate, however, was completely abolished by azide. Therefore, it seems likely that a functional nitrate reductase system, rather than nitrate, controls the formation of the enzymes repressible by nitrate.     

Regulation of reductase formation in Proteus mirabilis

Summary Prteus mirabilis can form four reductases after anaerobic growth: nitrate reductase A, chlorate reductase C, thiosulfate reductase and tetrathionate reductase. The last three enzymes are formed constitutively. Nitrate reductase is formed only after growth in the presence of nitrate, which causes repression of the formation of thiosulfate reductase, chlorate reductase C, tetrathionate reductase and hydrogenase. Formic dehydrogenase assayed with methylene blue as hydrogen acceptor is formed under all conditions.Two groups of chlorate resistant mutants were obtained. One group does not form the reductases and formic dehydrogenase. The second group does not form nitrate reductase, chlorate reductase and hydrogenase, but forms formic dehydrogenase and small amounts of formic hydrogenlyase after growth without hydrogen acceptor or after growth in the presence of thiosulfate or tetrathionate. Nitrate prevents the formation of formic dehydrogenase, thiosulfate reductase and tetrathionate reductase in this group of mutants. Only after growth with thiosulfate or tetrathionate the reductases for these compounds are formed. Anaerobic growth of the wild type in complex medium without a fermentable carbon source is strongly stimulated by the presence of nitrate. Tetrathionate and thiosulfate have no effect at all or only a small effect. The results show that in the presence of tetrathionate or thiosulfate the bacterial metabolism is fully anaerobic, as these cells also contain formic hydrogenlyase.     

Regulation of the Swarming Inhibitor disA in Proteus mirabilis

The disA gene encodes a putative amino acid decarboxylase that inhibits swarming in Proteus mirabilis. 5′ rapid amplification of cDNA ends (RACE) and deletion analysis were used to identify the disA promoter. The use of a disA-lacZ fusion indicated that FlhD₄C₂, the class I flagellar master regulator, did not have a role in disA regulation. The putative product of DisA, phenethylamine, was able to inhibit disA expression, indicating that a negative regulatory feedback loop was present. Transposon mutagenesis was used to identify regulators of disA and revealed that umoB (igaA) was a negative regulator of disA. Our data demonstrate that the regulation of disA by UmoB is mediated through the Rcs phosphorelay.     

Transposon mutagenesis in Proteus mirabilis.

A technique of transposon mutagenesis involving the use of Tn5 on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that Tn5 transposed in P. mirabilis at a frequency of ca. 4.5 x 10(-6) per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridizaiton to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed.     

Lipoprotein from Proteus mirabilis.

The biosynthesis of a Proteus mirabilis outer membrane protein of molecular weight of approximately 7,000 was found to be relatively resistant to puromycin and rifampin, as is the case for the Escherichia coli liporotein. Furthermore, the existence of the lipoprotein in P. mirabilis was indicated by a comparison of the amino acid compositions of the purified free and bound forms of this protein with those of the E. coli free and bound lipoproteins.     

Fumarate reduction in Proteus mirabilis.

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorate-resistant mutant strains of the wild type, fumarate reductase appeared to be affected. 2. Cytoplasmic membrane suspensions isolated from anaerobically grown P. mirabilis oxidized formate and NADH with oxygen and with fumarate, too. 3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochrome b, cytochrome a1 and cytochrome d. Cytochrome b was reduced by NADH as well as by formate to approximately 80%. 4. 2-n-Heptyl-4-hydroxyquinilone-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state. 5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochrome b. 6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen. 7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochrome b as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grown P. mirabilis is presented.     

Core region in Proteus mirabilis lipopolysaccharide.

Four R mutants of P. mirabilis were isolated. The composition of their degraded polysaccharides (PS) obtained from the respective lipopolysaccharides (LPS) as well as the composition and properties of the PS-fractions separated by column chromatography were examined. The results were compared with those obtained with PS of the wild type. One of the mutants could be classified as an Ra-type mutant, presenting a complete LPS core. This polysaccharide core contains: galacturonic acid, glucosamine, glucose, D-glycero-D-mannoheptose, L-glycero-D-mannoheptose in a molar ratio of 1 : 1 : 1 : 1 : 2 and 2-keto-3-deoxyoctonate. Taking into consideration the common sugars described previously in the LPS chemotypes of P. hauseri, the composition of the complete core region mentioned above represents the LPS core part of all the chemotypes, containing two different heptoses.     

Endonuclease from Proteus mirabilis

Two isoforms of nuclease displaying DNase and RNase activities were found in the culture liquid and periplasm of Proteus mirabilis. The enzyme was isolated from the periplasm and then purified to a functionally homogeneous state. The nuclease was equally potent in cleaving denatured and native DNAs by the endonuclease mechanism and was designated Pm endonuclease. The endonuclease was shown to be a temperature-dependent enzyme with a pH optimum of 10.4-10.6, requiring the presence of bivalent metal ions and inhibited by citrate and ethylenediaminetetraacetate.     

Endonuclease from Proteus mirabilis

Two isoforms of nuclease displaying DNase and RNase activities were found in the culture liquid and periplasm of Proteus mirabilis. The enzyme was isolated from the periplasm and then purified to a functionally homogeneous state. The nuclease was equally potent in cleaving denatured and native DNAs by the endonuclease mechanism and was designated Pm endonuclease. The endonuclease was shown to be a temperature-dependent enzyme with a pH optimum of 10.4–10.6, requiring the presence of bivalent metal ions and inhibited by citrate and ethylenediaminetetraacetate.     

Phosphatase from Proteus mirabilis

Cell-free preparations of Proteus mirabiliscontained a phosphatase (EC 3.1.3.1) whose activity surpassed that of alkaline phosphatase from Escherichia coli. Phosphatase was also found in the culture liquid of P. mirabilis. The composition of proteins displaying enzyme activity was assayed by polyacrylamide gel electrophoresis. Enzyme synthesis was studied at various stages of bacterial growth. Biosynthesis of phosphatase in P. mirabilis(similarly to that found in other bacteria) was shown to be induced under conditions of inorganic phosphate deficiency in the medium.