beta-Ketoadipate pathway in Trichosporon cutaneum modified for methyl-substituted metabolites.

Trichosporon cutaneum, when grown with p-cresol, catalyzed intradiol fission of the benzene nucleus of 4-methylcatechol before the complete catabolism of these two substrates. Steps in their conversion to pyruvate and acetyl coenzyme A were investigated by using cell extracts, and some properties of various new microbial catabolites are also described. These included (-)-2,5-dihydro-3-methyl-5-oxofuran-2-acetic acid (beta-methylmuconolactone) and (-)-3-keto-4-methyladipic acid and its coenzyme A ester; the latter was degraded by an enzymatic reaction sequence that included the coenzyme A esters of methylsuccinic, itaconic, and citramalic acids. A notable feature of this sequence is the formation of beta-methylmuconolactone which can be readily metabolized, in contrast to the analogous reaction in bacteria that gives the "dead-end" compound gamma-methylmuconolactone; this compound cannot be enzymatically degraded and so renders the beta-ketoadipate pathway unavailable for methyl-substituted bacterial sources of carbon that are catabolized by way of 4-methylcatechol.     

Nucleotide sequence of the gene determining plasmid-mediated citrate utilization.

The citrate utilization determinant from transposon Tn3411 has been cloned and sequenced, and its polypeptide products have been characterized in minicell experiments. The nucleotide sequence was determined for a 2,047-base-pair BglII restriction endonuclease fragment that includes the citrate determinant. This region contains an open reading frame that would encode a 431-amino-acid very hydrophobic polypeptide and which is preceded by a reasonable ribosomal binding site. However, the single polypeptide found in minicell experiments had an apparent molecular weight of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Genetic analysis of Escherichia coli oligopeptide transport mutants.

The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.     

Generation of a large, protonophore-sensitive proton motive force and pH difference in the acidophilic bacteria Thermoplasma acidophilum and Bacillus acidocaldarius.

The mechanism by which acidophilic bacteria generate and maintain their cytoplasmic pH close to neutrality was investigated. For this purpose we determined the components of proton motive force in the eubacterium Bacillus acidocaldarius and the archaebacterium Thermoplasma acidophilum. After correction for probe binding, the proton motive force of untreated cells was 190 to 240 mV between external pH 2 and 4. Anoxia diminished total proton motive force and the transmembrane pH difference by 60 to 80 mV. The protonophore 2,4-dinitrophenol abolished the total proton motive force almost completely and diminished the transmembrane pH difference by at least two units. However, even after correction for probe binding, protonophore-treated cells maintained a pH difference of approximately one unit.     

Locations of hook-associated proteins in flagellar structures of Salmonella typhimurium.

Hooks of the flagella of Salmonella typhimurium were purified from an flaL mutant. Hook-associated proteins, namely HAP1, HAP2, and HAP3, were separated from them, and the antibody against each HAP was prepared. By immunoelectron microscopic observation, these three kinds of antiHAP antibodies were found to bind on the distal ends of hooks of filamentless mutants consistently with their composition of HAPs. The antiHAP2 antibody bound to the very tops of the claw-shaped ends of the hooks which contain all three HAPS. The antibodies against HAP1 and HAP3 bound to the basal areas and the middle areas, respectively, of the claw-shaped ends. The order of disassembly of the component proteins by heat treatment of the hook structure from the filamentless mutants was (HAP2, HAP3) greater than HAP1 greater than hook protein. These observations were consistent with our layered structure model: HAP1, HAP3, and HAP2 are assembled at the distal end of the hook in this sequence. All three HAPs were detected in the hook-filament complexes prepared from a flagellate strain. When the hook-filament structure was treated with antibody against HAP1 and with the anti-rabbit immunoglobulin G antibody, the antibody aggregate was observed in the region corresponding to the boundary between filament and hook. This observation strongly suggests that HAP1 is the protein connecting filament with hook. The locations of HAP2 and HAP3 in the hook-filament structure were not clarified with the same procedure.     

Immunological investigation of the distribution of cytochromes related to the two terminal oxidases of Escherichia coli in other gram-negative bacteria.

Monospecific antibodies were raised against the two terminal oxidase complexes of the aerobic respiratory chain of Escherichia coli. These are the cytochrome d and cytochrome o complexes. The antibodies were used to check for the occurrence of cross-reactive antigens in membrane preparations from a variety of gram-negative bacteria by rocket immunoelectrophoresis and immunoblotting techniques. With these criteria, proteins closely related to the cytochrome d complex of E. coli appeared to be widely distributed. Among the strains containing cytochrome d-related material were Serratia marcescens, Photobacterium phosphoreum, Salmonella typhimurium, Klebsiella pneumoniae, and Azotobacter vinelandii. The data suggest that the d-type terminal oxidase in many of these strains is associated in a complex with b-type and a1-type cytochromes, as has been found to be the case in E. coli. K. pneumoniae and S. typhimurium were also shown to have material cross-reactive to the E. coli cytochrome o complex.     

Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system.

The enzymes for luminescence in Vibrio fischeri are induced by the accumulation of a species-specific metabolite (autoinducer) in the culture medium. Tritium-labeled autoinducer was used to study the mechanism of autoinduction. When 3H-autoinducer was added to suspensions of V. fischeri or Escherichia coli, cellular concentrations equaled external concentrations. For V. fischeri, equilibration of 3H-autoinducer was rapid (within 20 s), and greater than 90% of the cellular tritium remained in unmodified autoinducer. When V. fischeri or E. coli cells containing 3H-autoinducer were transferred to autoinducer-free buffer, 85 to 99.5% of the radiotracer escaped from the cells, depending on the strain. Concentrations of autoinducer as low as 10 nM, which is equivalent to 1 or 2 molecules per cell, were sufficient for induction, and the maximal response to autoinducer occurred at about 200 nM. If external autoinducer concentrations were decreased to below 10 nM after induction had commenced, the induction response did not continue. Based on this study, a model for autoinduction is described wherein autoinducer association with cells is by simple diffusion and binding of autoinducer to its active site is reversible.