Bacteriophages of Clostridium saccharoperbutylacetonicum

The morphological properties of the twelve previously described HM-phages were examined by electron microscopy. Specimens were prepared by air-drying and shadow-casting method using purified phage suspensions. As a result, the HM-phages were classified into three morphologically distinct groups, 1, 11 and 111. Group 1 phages were HM 1, HM 2, HM 8, HM 9, HM 10, HM 11 and HM 12. These phages had a spherical head about 100 mμ in diameter and a rudimentary tail. Group 11 phages were HM 3, HM 4, HM 5 and HM 6. These phages had a spherical head about 100 mμ in diameter and a tail with contractile sheath, and the normal tail of these phages was about 100 mμ in length, and the contracted sheath was about 50 mμ in length, Group 111 phage was HM 7 alone. This phage had a spherical head about 120 mμ in diameter and a relatively long tail about 350 mμ in length.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The fine structure of phage HM 2 (group I) active on Clostridium saccharoperbutylacetonicum was studied by an electron microscopy with a negative-staining technique, and compared with those of more conventional types, phages HM 3 (group II) and HM 7 (group III), whose tails were clearly observed by a shadow-casting technique. This study revealed that phage HM 2 had an intricate tail which was not observed by a shadow-casting technique.

Phage HM 2 has an icosahedral head about 450 Å in diameter and a non-contractile tail about 300 Å long. The distal 130 Å of the tail axis has a width of 80 Å which is wider than the upper portion of the tail (50 to 60 Å). The distal enlargement is not seen in the hollow tail. Twelve fibrous-shaped appendages are attached symmetrically at the upper portion of tail axis and extend toward the distal base of the tail. Their length is a little shorter than 300 Å. They combine with divalent cations in the phage dilution medium, and also adsorb the host cell debris.

Phage HM 3 has an icosahedral head about 770 Å in diameter and a tail about 1000 Å long and 150 Å wide with contractile sheath. Phage HM 7 has an icosahedral head about 750 Å in diameter and a long non-contractile tail about 2000 Å long and about 120 Å wide with forked tip.

The structure of the tail of phage HM 2 is quite different from those of phages HM 3 and HM 7 hitherto described and those of the various phages of other bacteria.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

Antiphage sera were produced in rabbits against the HM-phages of Clostridium saccharoperbutylacetonicum; on the basis of cross-neutralization experiments with homologous and heterologous antisera, the twelve HM-phages were classified into three serological groups, termed I, II and III. Group I contained seven phages, i.e., HM 1, HM 2, HM 8, HM 9, HM 10, HM 11 and HM 12. Group II contained four phages, i.e., HM 3, HM 4, HM 5 and HM 6, and group III one phage, i.e., HM 7. This classification was in accord with morphological one that was reported in the preceding paper. By using the K value of antisera, the degree of serological relatedness among the phages within groups I and II was demonstrated. On the bases of serological similarities and of dissimilarities in host-rang specificity, the phages of groups I and II are considered as host range mutants derived from an identical ancestor, HM 1 and HM 3, respectively.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

Further characterization of the HM-phages of Clostridium saccharoperbutylacetonicum was described; plaque morphology, thermal inactivation, pH stability, inactivation by ultraviolet irradiation, and 1ysis of infected culture. Differences in the characteristics were observed among the three groups of HM-phages.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The HM-phages contained only deoxyribonucleic acid (DNA) as the nucleic acid moiety. The DNA was extracted from the phages by the phenol method. The content of guanine plus cytosine (%G + C) in the DNA was determined by paper chromatography and by thermal denaturation method. The values of HM 2 (group I), HM 3 (group II) and HM 7 (group III) were 35, 30 and 29, respectively.

The DNA was also isolated from the two host strains of Clostridium saccharoperbutylacetonicum by the method of Marmur and by Saito and Miura’s phenol extraction method. The %G + C of the DNA was 31. No unusual bases were detected in either the bacterial or phage DNA.     

Bacteriophages of Clostridium saccharoperbutylacetonicun

Some characteristics of the twelve phages were given. Phages were obtained from the twelve abnormal broths in the industrial production of acetone and butanol by use of Cl. saccharoperbutylacetonicum, producing a high proportion of butanol. These new phages were designated as HM and numbered consecutively in the order of appearance. The HM-phages were highly specific for the strains of this bacterium. Each phage could be distinguished from the others by its differential host specificity against various phage-resistant mutants of this bacterium. The HM-phages were not temperate ones under our experimental conditions. They were divided into two groups on the basis of their stability in the salts solution. While one group was slightly less stable in 0.85 percent saline or 0.067 m phosphate buffer, the other was strikingly unstable. The addition of magnesium ion was effective for increase in stability of both groups.

The double-layer method similar to that described by Adams, with modifications to incubate the plates anaerobically, was applied to assay the phages of butanol-producing bacteria, Cl. saccharoperbutylacetonicum, after the studies on some factors influencing the plaque formation. Factors influencing the number and size of plaque were as follows; agar concentration and amount of overlayer medium, pH of media, age and number of host cells, temperature and period of incubation, and so on. Plaques formed by this method were medial size and easily counted. Assay of viable cells of this anaerobic bacterium was also possible by this method with slight modifications.     

Antitumor Activity of an Alkali Extract of Clostridium saccharoperbutylacetonicum Cells

Bacillus thuringiensis var. israelensis produces 130-kDa proteins which are toxic to mosquito larvae. The ISRH4 gene encoding 1,180 amino acids of the 130-kDa insecticidal protein was fused with lac Z′ on a plasmid, pUC19, and sequentially deleted from the C-terminus to construct a series of deletion mutants. All the deletion mutant genes directed the production of truncated ISRH4 proteins fused with the α-complementing fragment of β-galactosidase in Escherichia coli cells in the presence of isopropyl β-d-thiogalactopyranoside. Analysis of the mosquito larvicidal activity of deletion mutant proteins revealed that the N-terminal 29 amino acids and the C-terminal 485 amino acids could be removed without loss of the activity.     

Sucrose-induced Autolysis and Development of Protoplast-like Cells of Clostridium saccharoperbutylacetonicum

A growing culture of Clostridium saccharoperbutylacetonicum partially lost its turbidity in the presence of 0.3 to 0.6 m sucrose without any extraneous supplements for cell wall degradation. The maximum effect was shown at 0.35 m of sucrose and the culture lost 40 to 50% of initial turbidity. The rate of lysis depended on the age of culture. The most rapid lysis occurred in the organisms of early exponential growing cultures, but no lysis was observed on those of late exponential and stationary phase cultures. The optimal pH was 5.5 to 6.0, and the optimal temperature 30 to 35°C. The sucrose-induced lysis was inhibited by bivalent cations (such as Ca²⁺, Mg²⁺), heavy metal cations (such as Cu²⁺, Pb²⁺), enzymic inhibitors (such as PCMB) and fixative agents (such as formalin, glutaraldehyde), while organisms whose growth had been inhibited by antibiotics (such as chloramphenicol, tetracycline) were also resistant to sucrose-induced lysis. The sucrose-induced lysis was accompanied by striking morphological conversion from original rod cells (3.0~6.0}0.4~0.6 μ) to spherical cells (1.0~ 1.2 μ diameter). The sucrose-induced lysis was also observed on the relative strains of C. saccharoperbutylacetonicum and C. sporogenes, but not observed on many other species of Clostridium and aerobic bacteria tested. It was suggested that sucrose-induced lysis was a kind of bacterial autolysis which was induced by sucrose treatment. The bacterial spheres developed during the lysis may be the protoplasts.     

Inducible Phage Tail-like Particles of Clostridium saccharoperbutylacetonicum and Its Related Strains

Two new kinds of high molecular bacteriocin, named as clostocins O and M, were found in Clostridium saccharoperbutylacetonicum and its related strains. Production of both clostocins was inducible by mitomycin C (MC) or ultraviolet ray. The active clostocins appeared in the bacterial cells at about 105 min after MC-treatment, increased rapidly during next 75 min and then released into the medium with the cell lysis. The clear lysis of O- and M-producing cells was observed at 3.5 hr and 4 hr respectively after MC-treatment. Clostocin O killed M-producing strains, and clostocin M did O-producing strains. Electron microscopic observation revealed that clostocins O and M were phage tail-like particles with contractile sheath round a core. The particles resembled some pyocins and also the tail of phage HM 3 of Cl. saccharoperbutylacetonicum.

It was also found that M-producing strains had simultaneously the ability of production of low molecular bacteriocin, named as clostocin D.     

The relationship between hydrogen gas and butanol production by Clostridium saccharoperbutylacetonicum

Two simultaneous fermentations were performed at 26 degrees C with simultaneous inocula using Clostridium saccharoperbutylacetonicum. Fermentation 1 prevented the gas formed by the biomass from escaping the fermentor while 2 allowed the gas formed to escape. Fermentor 1 provided for the production of butanol, acetone, and ethanol, while when the H(2) formed was allowed to escape with fermentor 2, neither butanol nor acetone were produced. Ethanol was also formed in both fermentors and began along with the initial growth of biomass and continued until the fermentations were complete. Butanol and acetone production began after biomass growth had reached a maximum and began to subside. The butanol-acetone-ethanol millimolar yields and ratios were 38:1:14 respectively. The fermentor 2 results show that a yield of 2.1 L H(2), 93 or 370 mmol H(2)/mol glucose, was formed only during the growing stage of growth; neither butanol nor acetone were produced; ethanol was formed throughout the fermentation, reaching a yield of 15.2 mmolar. It appears that hydrogen gas is required for butanol production during the resting stage of growth.     

Morphological Changes during Conversion of Clostridium saccharoperbutylacetonicum to Protoplasts by Sucrose-Induced Autolysis

When exponentially growing cells of Clostridium saccharoperbutylacetonicum (ATCC 13564) were exposed to hypertonic concentrations of sucrose (0.3–0.5 M), rapid degradation of the cell wall occurred (sucrose-induced autolysis). The morphological changes from the original rod-shaped cells to protoplasts during the sucrose-induced autolysis were investigated by phase contrast and electron microscopy. When the cells were autolysed in the sucrose solution (0.35 M), each cell began to swell at the middle or at one pole and then formed a small bulb at the swollen part. The bulb consisted of the cytoplasm which was enveloped by the plasma membrane and extruded from the small gap produced by the degradation of the cell wall. The bulb gradually enlarged as lysis progressed, and finally became a protoplast which had no cell wall. The large pre-division cell frequently formed the bulb at the middle (septal site), while the small post-division cell formed the bulb at the pole.     

Mode of Action of Clostridium Phage HM 7-Induced Lytic Enzyme on Clostridium saccharoperbutylacetonicum Cell Wall Peptidoglycan

The action of Clostridium phage HM 7-induced lytic enzyme on the cell wall peptidoglycan of Clostridium saccharoperbutylacetonicum was investigated. The cell wall peptidoglycan of this strain contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00: 2.08: 0.97; 0.92: 0.68. It was strongly digested when incubated with the lytic enzyme. This digestion was accompanied by the release of NH₂-terminal l-alanine without a concomitant release of COOH-terminal amino acids and reducing groups. Chromatography of the lytic enzyme digest resulted in only two fractions, each of which was chromatographically homogeneous. One was a polysaccharide consisting of glucosamine and muramic acid in molar ratios 1.00: 0.78, and other was a peptide composed of glutamic acid, alanine and diaminopimelic acid in molar ratios of 1.00: 2.09: 1.05. These results indicate that phage HM 7-induced lytic enzyme is N-acetylmuramyl-l-alanine amidase, which cleaves the linkage between N-acetylmuramic acid and l-alanine.

A possible structure for the cell wall peptidoglycan was also proposed.     

Isolation and Characterization of Temperate Bacteriophages of Clostridium difficile

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages C2, C5, C6, and C8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of C2 showed nucleotide homology to the sequenced C. difficile strain CD630.     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

Purification and Properties of Phage HM 7-Induced Lytic Enzyme of Clostridium saccharoperbutylacetonicum

A lytic enzyme was isolated from phage HM 7-induced lysate of Clostridium saccharoperbutylacetonicum, and purified about 200-fold by precipitation with ammonium sulfate, gel filtration with Sephadex G–75 and ampholine isoelectric focusing. The purified lytic enzyme had an apparent homogeneity on disc-electrophoresis, and the character of acidic protein showing isoelectric point at pH 4.0. The molecular weight of lytic enzyme was estimated to be about 100,000 from the result of SDS-polyacrylamide gel electrophoresis. The optimum pH for the lytic enzyme activity was 6.5. Maximum activity occurred at 30 to 35°C, and at the ionic strength of 0.04 m or above. The lytic enzyme activity was stimulated about 140% by 10?³ m EDTA. The lytic enzyme lysed the living cells, but it had a narrow specificity which was restricted to a certain species of Clostridium such as Cl. saccharoperbutylacetonicum, Cl. butyricum, Cl. botulinum, Cl. sporogenes, and Cl. thiaminolyticum.     

Comparative investigations of growth and solvent formation in ‘Clostridium saccharoperbutylacetonicum’ DSM 2152 and Clostridium acetobutylicum DSM 792

The butanol and acetone-producing strain DSM 2152, invalidly described as ‘Clostridium saccharoperbutylacetonicum’ is compared with the type strain C. acetobutylicum, DSM 792, with respect to solvent and acid formation at varying pH values and growth rates. Batch cultures, product-limited chemostat and pH-auxostat cultures were used for characterization. Under all conditions strain DSM 2152 produced much lower amounts of butyric and acetic acids than the type strain. The pH optimum for solvent formation was higher, ie 5.5 instead of 4.5. Solvent formation occurred at higher dilution rates, but below 0.1 h⁻¹ a lower solvent concentration was obtained, indicating that acid production was too low to provide a sufficient amount for acetone formation. The results are discussed in the light of recent publications on the taxonomy of butanol-acetone producing clostridia using 16S rRNA sequence analysis and other nucleic acid data. The presently suggested ‘phylogenetic’ classification of the collective species, C. acetobutylicum, is also reflected in the fermentation characteristics. Received 21 December 1998/ Accepted in revised form 22 January 1999