Physiological Effects of a Constitutive Tryptophanase in Bacillus alvei.

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological effects of a constitutive tryptophanase in Bacillus alvei. J. Bacteriol. 90:604-610. 1965.-Tryptophanase synthesis in B. alvei is not under the control of tryptophan and is not subject to catabolite repression. Exogenously supplied tryptophan was converted to indole by tryptophanase, and was excreted into the culture medium. The amount of indole excreted was dependent upon the concentration of tryptophan supplied. At intermediate levels of tryptophan (5 to 15 mug/ml), the excreted indole was completely reutilized by the cell, in contrast to the result with higher levels. Indole reutil zation was shown to be dependent upon a functional tryptophan synthetase. In the absience of exogenous tryptophan, indole was excreted into the culture medium at an earlier physiological age. The early indole was shown not to be a consequence of tryptophanase action. The early indole accompanied uniformly the normal process of tryptophan biosynthesis, and the fission of indole-3-glycerol phosphate was suggested as the origin of the excreted indole.     

Catalytic studies on tryptophanase from Bacillus alvei

Tryptophanase from Bacillus alvei exhibited the expected spectrum of pyridoxal-5'-phosphate-dependent reactions. It exhibited l-serine dehydratase, S-alkyl-cysteine lyase, and cysteine desulfhydrase activities, as well as the classic tryptophanase reactions (all beta elimination reactions). It also acted as a tryptophan synthetase (beta replacement reactions) using indole plus l-serine or l-cysteine or S-methyl-cysteine as substrates. The beta elimination reactions are simple competitors of the replacement reactions for the same amino acid substrates. The kinetics of the reactions were examined in detail using a coupled continuous spectrophotometric assay. A product (indole) inhibition study of the beta elimination reaction with tryptophan showed simple, noncompetitive inhibition; the same study with allosubstrates showed noncompetitive inhibition by indole. These product studies provided data on the beta replacement reactions as well. The results are discussed in terms of a mechanism for the B. alvei tryptophanase.     

Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological role of tryptophanase in control of tryptophan biosynthesis in Bacillus alvei. J. Bacteriol. 91:667-672. 1966.-Indole excretion occurred early in the exponential growth phase, and derived mainly from biosynthetic intermediates of tryptophan. Tryptophan cleavage by tryptophanase contributed about 1.5% of the indole excreted. In the presence of exogenous tryptophan (5 to 10 mug/ml), excretion of early indole was not observed. Experiments with isotopically labeled indole and tryptophan showed that a low rate of endogenous tryptophan biosynthesis occurred constantly during growth. Both exogenously and endogenously supplied tryptophan were degraded by tryptophanase. As a consequence, the intracellular tryptophan concentration appeared to be maintained at a constant low level. It was suggested that the action of tryptophanase is an example of an enzymatic mechanism which controls the level of a specific metabolite pool.     

Anthrose biosynthetic operon of Bacillus anthracis

The exosporium of Bacillus anthracis spores consists of a basal layer and an external hair-like nap. The nap is composed primarily of the glycoprotein BclA, which contains a collagen-like region with multiple copies of a pentasaccharide side chain. This oligosaccharide possesses an unusual terminal sugar called anthrose, followed by three rhamnose residues and a protein-bound N-acetylgalactosamine. Based on the structure of anthrose, we proposed an enzymatic pathway for its biosynthesis. Examination of the B. anthracis genome revealed six contiguous genes that could encode the predicted anthrose biosynthetic enzymes. These genes are transcribed in the same direction and appear to form two operons. We introduced mutations into the B. anthracis chromosome that either delete the promoter of the putative upstream, four-gene operon or delete selected genes in both putative operons. Spores produced by strains carrying mutations in the upstream operon completely lacked or contained much less anthrose, indicating that this operon is required for anthrose biosynthesis. In contrast, inactivation of the downstream, two-gene operon did not alter anthrose content. Additional experiments confirmed the organization of the anthrose operon and indicated that it is transcribed from a sigma(E)-specific promoter. Finally, we demonstrated that anthrose biosynthesis is not restricted to B. anthracis as previously suggested.     

Purification of an extracellular thiol-dependent hemolysin from Bacillus alvei]

Alveolysin a sulfhydryl-dependent cytolytic extracellular protein released by Bacillus alvei has been purified by salting-out by ammonium sulfate, gel filtration, isoelectric focusing on pH gradient and chromatography on DEAE-cellulose. The purified protein after reduction by thiols (active hemolytic form) proved homogeneous by disc polyacrylamide gel electrophoresis and by gel immunodiffusion. The molecular weight was 60,000 daltons, Two molecular forms of pI 5.1 and 7.0 were detected by gel isoelectrofocusing. The toxin was lethal to the mouse. Lytic activity was inhibited by cholesterol and antistreptolysin O anstisera. Immunological cross-reaction was observed between alveolysin and streptolysin O.     

Physiological studies on Phymatotrichum omnivorum. IX. Synthesis of indole acetic acid in vitro.

The cotton root rot fungus, Phymatotrichum omnivorum, synthesized indole acetic acid (IAA) in culture medium containing tryptophan as the only source of nitrogen. Synthesis of IAA was not affected by illumination, and maximum amounts of the auxin were produced at 30 days. The optimum hydrogen ion concentration and temperature for IAA production were 6.5 and 28 degrees C, respectively. Indole ethanol (IEA) was also detected in tryptophan media, primarily during the active growth of the organism.     

Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.     

Proposed pathway for biosynthesis of the S-layer glycoprotein of Bacillus alvei.

The outer surface of the murein sacculus of the eubacterium Bacillus alvei is covered by a surface layer (S-layer) glycoprotein. The glycan chain of this S-layer glycoprotein consists of trisaccharide repeating units with ManNAc, Gal, and Glc as constituents. From cell extracts of B. alvei, nucleotide-activated derivatives of ManNAc, Gal, Glc, and GlcNAc were isolated. Furthermore, GDP- and dolichyl-activated oligosaccharides were obtained. On the basis of the isolated putative glycoprotein precursors, a pathway for the biosynthesis of the oligosaccharide chain is proposed.     

Substrate-protein interaction in tryptophanase from Bacillus alvei. Kinetic and spectral evaluations.

This investigation studied the substrate protein interaction of the alpha, beta elimination reaction in tryptophanase (EC 4.1.99.1). The results of this work are 2-fold. (a) The presence of multiple enzyme sites was found to be related to the observed kinetic patterns of inhibition. Indole analogues caused competitive inhibition in the tryptophanase reaction and noncompetitive inhibition in the dehydratase reaction. Inhibition patterns of alanine for these activities were reserved. (b) Under some conditions, compounds which bind presumably at the indole site modified the spectral and fluorescent characteristics of the enzyme. The addition of anthranilate to the enzyme resulted in a broad absorption band around 350 nm. This absorption band was distinct from that formed by alanine addition. Based on absorption data, both of these compounds could be bound simultaneously. The optical activity of tryptophanase was reported for the first time. Indole analogues caused greater conformational alterations in the circular dichroism spectra than 3-carbon analogues. The calculated anisotrophy factors, as well as fluorescent quenching data, suggest a more direct interaction between indole analogues and pyridoxal-P than between 3-carbon compounds that the coenzyme. It is proposed that the indole site is the dominant recognition site. The data are consistent with the three-dimensional aspects of space-filling models of Schiff's bases evaluated in terms of multiple site binding.     

Molecular cloning and characterization of Bacillus alvei thiol-dependent cytolytic toxin expressed in Escherichia coli

A chromosomal DNA fragment from Bacillus alvei, encoding a thiol-dependent haemolytic product known as alveolysin (Mr 60,000, pI 5.0) was cloned in Escherichia coli SK1592, using pBR322 as the vector plasmid. Only a single haemolysin-positive clone was identified, by testing for haemolysis on blood agar plates. The haemolytic material was associated with the host bacterial cell. It was released by ultrasonic disruption and purified 267-fold. A 64 kDa polypeptide of pI 8.2 cofractionated with haemolytic activity during gel filtration chromatography and isoelectric focusing. It behaved identically to alveolysin in its activation by thiols, inactivation by thiol group reagents, inhibition by cholesterol, and neutralization, immunoprecipitation and immunoblotting by immune sera raised against alveolysin and streptolysin O.