Palmer Rogers

Genetics and Biochemistry of Clostridium Relevant to Development of Fermentation Processes

Publisher Summary This chapter focuses on those areas of research or directions in the study of the biology of the clostridia that seem to be of the highest priority for the immediate future. First, it is with respect to the biochemistry of clostridia fermentations. Second, in the realm of infant genetics of the clostridia it is not hard to find areas of research that are essential. Third, the area of clostridial enzyme complexes that degrade biopolymers should be of great future interest. Fourth, research on morphogenesis and endospore formation in the clostridia should reveal similar mechanisms of global regulatory changes as shown in the aerobic bacilli. This knowledge seems important to an understanding of the major internal mechanisms and environmental signals controlling solvent production in C. acetobutylicum . The prospect of utilizing the clostridia in any cost-effective process in the future will depend heavily upon improving their tolerance to toxic solvent and acid products. Research in this area is still very rudimentary. With the new tools of genetics applied to this problem in the clostridia, perhaps some progress in the understanding of tolerance will be made.

Studies in management of the contaminated wound: I. Technique of closure of such wounds together with a note on a reproducible experimental model

Abstract A readily reproducible experimental wound infection model has been developed to assess the determinants of infection in primarily closed contaminated wounds. The needle puncture tract, the suture material, the diameter and quantity of suture remaining within the wound, and the tightness with which each suture is tied have been identified as factors which enhance the inflammatory response of the contaminated wound to infection. The increased likelihood of infection attending primary closure of contaminated wounds with percutaneous sutures gives strong support to the thesis that percutaneous sutures should be avoided in the closure of contaminated wounds.

Mechanism of canavanine death in Escherichia coli: II. Membrane-bound canavanyl-protein and nuclear disruption

Abstract Electron microscopy of canavanine-grown Escherichia coli demonstrates two profound changes in intracellular organization. First, two to eight large electrondense bodies are found attached to the cell membrane. These canavanineinduced bodies have been isolated from the cells and shown to have an irregular, amorphous structure with pieces of cell membrane attached to them. Second, extensive physical disruption of the nuclear region is observed in cells grown with canavanine under conditions that produce death. More than 70% of the canavanyl-protein formed is attached to the heavy cell fraction containing the canavanine-induced bodies; they are largely protein in nature, and their amino acid composition is similar to that of total E. coli protein except that canavanine replaces most of the arginine. A canavanine-resistant mutant of an arginine auxotroph of E. coli is described. This mutant incorporates canavanine into protein but it remains viable in the presence of 1 mg/ml. of l -canavanine, and can subsequently produce bacteriophage upon T4 infection. Electron micrographs of this mutant show the presence of the canavanine-induced bodies, but the nuclear region remains intact. Our results suggest that the lethal action of l -canavanine involves incorporation of this analog into membrane-bound protein(s) that interrupts the normal organization and functioning of the E. coli genome.

Mechanism of canavanine death in Escherichia coli: I. Effect of canavanine on macromolecular synthesis☆

Abstract Canavanine-dead Escherichia coli are unable to support the growth of T-even bacteriophage. Loss of the ability to make virus is directly related to the kinetics of canavanine death and is due to the synthesis of host protein containing canavanine. T4-infected canavanine-dead cells did not form phage-induced messenger RNA and consequently no phage-induced protein and DNA synthesis were observed. Amino acid incorporation into protein by cell-free extracts indicated a defect in the protein synthesizing system of canavanine-dead cells. It is shown that the washed ribosomes from dead cells are only slightly active while the soluble fraction is normal. Activity is restored when synthetic mRNA is supplied to ribosomes from cells rendered non-viable by canavanine. Thus, the protein synthesizing machinery in canavanine-dead E. coli appears intact and the low level of mRNA in cell extracts indicates that very little transcription takes place in the dead cells. Crude extracts from dead cells show high levels of RNA polymerase activity. However, plasmolyzed canavanine-dead cells show lowered levels of RNA synthesis in the presence of exogenous ribonucleoside triphosphates. DNA synthesis is not initiated in dead cells by supplying excess arginine followed by plasmolysis and the addition of deoxynucleoside triphosphates. Canavanine-dead cells retain the ability to oxidize glucose and generate ATP. These results are consistent with the hypothesis that, during canavanine death, proteins containing this amino acid analog are synthesized, which eventually block transcription of all DNA in the intact cell.

Analysis of Tn916-induced mutants of Clostridium acetobutylicum altered in solventogenesis and sporulation.

SummaryThe conjugative transposon Tn916 was used for mutagenesis ofClostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be, in the same region of the chromosome. These results predict, that multiple regulatory elements are required to induce solvent production and sporulation.

Effect of arginine and canavanine on arginine messenger RNA synthesis.

Summary The level of messenger RNA for the arg ECBH gene cluster in Escherichia coli was determined by hybridization of 3 H-pulse-labeled RNA with DNA from 80darg. L-Arginine repressed the level of hybridizable message 7 to 24 fold lower than that observed in arginine-deprived cells. Added L-canavanine had little effect on the level of hybridizable message during arginine-deprivation. Our data suggest that the arginine regulon is repressed at both the level of transcription and translation.

Metabolism of leptospirae: I. Utilization of amino acids and purine, and pyrimidine bases

Abstract The utilization of exogenous amino acids, purines, and pyrimidine by Leptospira pomona has been examined, with the following results: (1) Only limited growth and no sparing effect of a complete amino acid mixture minus asparagine was obtained when ammonia was omitted or limited in the growth medium. (2) The replacement by asparagine of the ammonium requirement was found to be due to the asparaginase activity of the rabbit serum additive in the medium. (3) Although amino acids supported only limited growth, the eight C 14 -labeled amino acids tested were incorporated into cellular protein and other cellular components. (4) Exogenous isoleucine, leucine, and valine were incorporated to an extent great enough to completely replace the incorporation of these endogenously synthesized amino acids into protein. However, the incorporation of exogenously supplied glutamate, and alanine was such that no significant diminution of these endogenously synthesized amino acids in protein was observed. (5) Purine bases were incorporated into L. pomona nucleic acids, and purine analogues were observed to inhibit growth. (6) Pyrimidine bases and carbamyl aspartate were not incorporated into L. pomona . Pyrimidine analogues had no effect on growth. (7) The results were discussed in connection with the peculiar requirement of ammonia for maximal growth of this heterotroph.

Isolation of a rapidly-sedimenting canavanyl-protein-DNA-membrane complex from Escherichia coli.

After growth of canavanine-sensitive strains of Escherichia coli with the amino acid analog, l-canavanine, a large portion of the cell DNA becomes bound to membrane-associated canavanyl-protein bodies. These rapidly-sedimenting complexes containing DNA and canavanyl-protein are detected by conversion of canavanine-killed bacteria to spheroplasts followed by gentle lysis and brief sedimentation through sucrose gradients. Electron microscopy of the isolated complex reveals that the canavanine-induced bodies are attached at multiple sites to high molecular weight DNA and pieces of bacterial membrane. Pronase or sodium lauryl sulfate release the DNA and canavanyl-protein from the complex. Neither cells grown without canavanine nor mutants resistant to the analog yield comparable complexes. Our results indicate that canavanine death is dependent upon the formation of canavanyl-protein-DNA-membrane aggregates and involves irreversible jamming of the E. coli genome at specific membrane sites.

Characteristics of butanol fermentation by a low-acid-producing Clostridium acetobutylicum B18

Fermentation characteristics of Clostridium acetobutylicum B18 were studied in batch experiments with and without pH control. This strain is shown to be potentially useful in simultaneous acetone-butanol-ethanol fermentation-separation systems because of its low acid production. In a pH-uncontrolled batch culture this strain produced mostly solvents, including 15 g/l of butanol. Ethanol production was low. Strain B18 recycled organic acids more efficiently than other strains. In particular, butyric acid was completely recycled when glucose was not limiting. Yield of liquid products (solvents plus organic acids) and carbon recovery in total products (gas plus liquid) were 33.1–36.4 wt% and 90–91 mol%, respectively, for 20–80 g/l of initial glucose. Glucose consumption and the percentage of butanol among solvents were higher at 32°C than at 37°C. Strain B18 required approximately 0.4 g/l of undissociated butyric acid at the onset of solvent production in pH-uncontrolled batch culture. The low undissociated butyric acid requirement enabled this strain to produce 13.8 g/l of butanol at a controlled pH of 6.0.

Regeneration of cells from protoplasts ofClostridium acetobutylicum B643

SummaryConditions that allow regeneration of cells fromClostridium acetobutylicum strain B643 protoplasts were studied. Protoplast formation and stabilization in minimal media with 50 mM CaCl2, 50 mM MgCl2 and 0.3 M sucrose were crucial to subsequent regeneration on soft yeast extract agar containing 25 mM CaCl2 and 25 mM MgCl2. A regeneration frequency of 8–25% was consistently obtained.