Killing Activity of Clostocin O

Clostocin O is a phage tail-like bacteriocin produced by Clostridium saccharoperbutylacetonicum Nl–4. One particle of clostocin O had an activity to kill one sensitive organism. Clostocin O had also the lytic activity, but this lytic activity was not an essential action of clostocin O, because clostocin O was able to show a sufficient killing activity even under the condition to inhibit its lytic activity. The biosynthesis of macromolecules (protein, RNA and DNA) in sensitive organisms was inhibited by clostocin O infection. The amounts of macro-molecules of the infected organisms were held at the initial level.     

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.     

Lytic Action of Inducible Bacteriocin Clostocin O

Clostocin O had a remarkable lytic action toward the exponentially growing organisms of Clostridium saccharoperbutylacetonicum No. 8. The cellular lysis was inhibited by addition of heavy metal cations such as Cu²⁺, Ni²⁺, and Cd²⁺, or fradiomycin and RNase, which had been reported to be the inhibitors for lytic enzymes such as some of clostridial phage-endolysin and clostocin O-endolysin. The formalin-treated organisms and antibiotic-treated organisms, of which autolysin activity was inhibited, were also lysed by clostocin O. The induced cellular lysis by clostocin O was thought to be due to the lytic enzyme attached to clostocin O.     

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.     

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.     

Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.     

Phage Receptor Material in Lactobacillus casei Cell Wall

A trichloroacetic acid (TCA)-soluble fraction, extracted using cold TCA, was derived from the cell wall of Lactobacillus casei strain S-1. It not only inhibited the adsorption of phage J-l but also desorbed these phages, in their active form, which had previously been adsorbed onto the cell walls. l-Rhamnose, one of the components of this TCA-soluble fraction, had an identical activity to this TCA-soluble fraction, on phage adsorption. This suggested that l-rhamnose is a part of phage receptor material in the cell wall of L. casei strain S-l; and the binding of the phage to the cell wall is reversible, even at 37 C.     

产朊假丝酵母细胞壁33 ku蛋白的功能研究

通过胰蛋白酶和枯草杆菌蛋白酶对产朊假丝酵母Candida utilis细胞壁的酶解,发现一种分子质量为33 ku的酵母细胞壁主要结构蛋白. 研究显示,在细胞壁上这种蛋白质与细胞壁绝大多数蛋白质成分不同, 它不被胰蛋白酶水解,但对枯草杆菌蛋白酶的作用敏感.33 ku蛋白存在于酵母菌整个对数生长期的细胞壁中,特别是在对数早期细胞壁中,它是唯一的对胰蛋白酶作用不敏感的蛋白质成分.实验证明,该蛋白质对维系酵母细胞壁骨架成分葡聚糖的相互连接和细胞壁的完整结构,具有重要作用,是一种重要的酵母细胞壁嵌合蛋白.     

Production of acetone,butanol and ethanol from palm oil waste by Clostridium saccharoperbutylacetonicum N1-4

Summary A study of the feasibility of utilizing palm oil waste as a substrate for acetonebutanol-ethanol fermentation by Clostridium saccharoperbutylacetonicum N1-4 and, hence, to reduce the biochemical oxygen demand (BOD) of waste disposal was carried out. Among the two types of palm oil waste tested, separator sludge was the better substrate and it was able to support production of solvents by C. saccharoperbutylacetonicum N1-4 without a need for any mineral supplements. Enzymatic hydrolysis catalyzed by cellulase prior to fermentation was found to increase the yield of butanol by 75% (from 2.47g/l to 4.37g/l) and decreased the BOD by 66% (from 26,500 ppm to 8,900 ppm).     

Purification and biological characterization of halocin C8, a novel peptide antibiotic from<Emphasis Type="Italic"> Halobacterium</Emphasis> strain AS7092

Halocins are bacteriocin-like proteins or peptides produced by many species of the family Halobacteriaceae. Halocin C8, excreted by the Halobacterium strain AS7092, is a single 6.3-kDa polypeptide with an isoelectric point of 4.4, which is sensitive to proteinase K but not to trypsin. Halocin C8 is quite stable, as it can be desalted, boiled, frozen, subjected to organic solvents, and stored in culture supernatant at 4°C or in dH₂O at –20°C for more than 1 year without losing activity. The purification of this halocin was achieved by combination of tangential flow filtration (TFF), Sephadex G50 and DEAE-sepharose chromatography. The N-terminal amino acid sequence was also determined by Edman degradation. Halocin C8 appeared to have a very wide activity spectrum, including most haloarchaea and even some haloalkaliphilic rods. When a sensitive strain of Halorubrum saccharovorum was exposed to halocin C8, the treated cells swelled at the initial stage, the cell wall appeared to be nicked and the cytoplasm was then extruded out, and the whole cell was eventually completely lysed. These results indicate that halocin C8 is a novel microhalocin and its primary target might be located in the cell wall of the sensitive cells.Communicated by W.D. Grant     

Killing of Gram-Negative Bacteria with Normal Human Serum and Normal Bovine Serum: Use of Lysozyme and Complement Proteins in the Death of Salmonella Strains O48

Serum is an environment in which bacterial cells should not exist. The serum complement system provides innate defense against microbial infections. It consists of at least 35 proteins, mostly in pre-activated enzymatic forms. The activation of complement is achieved through three major pathways: the classical, alternative, and lectin. Lysozyme, widely present in body fluids, catalyzes the hydrolysis of β 1,4 linkage between N-acetyloglucosamine and N-acetylmuramic acid in the bacterial cell wall and cooperates with the complement system in the bactericidal action of serum. In this study, ten strains of serotype O48 Salmonella, mainly associated with warm-blooded vertebrates and clinically important causing diarrhea in infants and children, were tested. The results demonstrated that the most efficient killing of Salmonella O48 occurred when all the components of normal bovine serum (NBS) and normal human serum (NHS) cooperated. To prove the role of lysozyme in the bactericidal activity of bovine and human serum, the method of serum adsorption onto bentonite (montmorillonite, MMT) was used. In order to investigate structural transitions accompanying the adsorption of serum components, we applied X-ray diffraction methods. The results of this investigation suggested that apart from lysozyme, other proteins (as, e.g., C3 protein or IgG immunoglobulin) were adsorbed on MMT particles. It was also shown that Ca²⁺ cations can be adsorbed on bentonite. This may explain the different sensitivities of the serovars belonging to the same O48 Salmonella serotype to NBS and NHS devoid of lysozyme.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The three representative HM-phages (HM 2, HM 3 and HM 7) of Clostridium saccharoperbutylacetonicum were used.

The adsorption rate of the phages HM 2 and HM 7 on the host bacteria was high, whereas that of the phage HM 3 was lower. The adsorption rates of the phages were maximum at pH 5.9~6.6, 30°C.

One-step growth experiment was successfully adapted to the phage-host systems of anaerobic bacteria by bubbling pure nitrogen gas into the medium in the growth tube. The growth characteristics of the HM-phages were investigated by using this technique. The minimal latent periods for phages HM 2, HM 3 and HM 7 were about 45, 90 and 120 minutes, respectively. The corresponding average burst sizes were approximately 500, 100 and 20, respectively. The growth of the phages was optimal at pH 6.2, 30~33°C. The phages failed to grow at 37°C, although the host bacteria multiplied at that temperature. By using a defined medium, it was found that calcium ion was not essential for the growth of the HM-phages.     

KRE genes are required for β‐1,6‐glucan synthesis,maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

The polysaccharide β‐1,6‐glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β‐1,6‐glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β‐1,6‐glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI‐anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β‐1,6‐glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.     

Aluminium speciation in the presence of wheat root cell walls: a wet chemical study

Hydrolysis of Al³⁺ was performed in the presence of isolated root cell walls from a series of wheat cultivars (Triticum aestivum L.) known to have differential tolerance to Al contamination. Aluminium speciation was dependent on the cell wall concentration. At low cell wall concentrations, significant amounts of the very toxic Al₁₃ species were formed. At higher cell wall concentrations, formation of the tridecamer was hindered or completely inhibited. The sensitive wheat cultivars displayed a higher affinity for aluminium than the tolerant cultivars. A possible Al tolerance mechanism based on cell wall permeability is discussed.     

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.     

AsSlt2基因对茄链格孢细胞壁完整性的调控

【背景】由茄链格孢(Alternaria solani)引起的马铃薯早疫病被普遍认为是马铃薯生产上的第二大叶部病害,在马铃薯各产区普遍发生,给马铃薯生产造成了巨大的经济损失。【目的】明确AsSlt2基因对茄链格孢细胞壁完整性的影响。【方法】在含有刚果红、细胞壁降解酶和十二烷基硫酸钠(sodiumdodecylsulfate,SDS)等细胞壁胁迫的培养基上观察ΔAsSlt2缺失突变株的生长情况,计算相对生长抑制率;通过实时荧光定量PCR (RT-qPCR)方法检测ΔAsSlt2菌株中细胞壁合成相关基因的表达情况;进一步检测ΔAsSlt2细胞壁中几丁质的含量及胞外酶活性。【结果】ΔAsSlt2缺失突变株对SDS、刚果红、细胞壁降解酶等细胞壁胁迫的敏感性增强,在加入细胞壁降解酶后突变株原生质体释放量显著增多;ΔAsSlt2对外源氧胁迫更敏感,突变株胞外过氧化物酶和漆酶活性均显著降低;进一步研究发现,ΔAsSlt2细胞壁中几丁质含量减少,几丁质合成相关基因与漆酶合成相关基因的表达量均明显降低。【结论】AsSlt2基因在茄链格孢细胞壁的完整性及抵御外界胁迫方面发挥重要作用。     

A cell wall-associated polysaccharide is required for bacteriophage adsorption to the Streptococcus thermophilus cell surface

Streptococcus thermophilus strain ST64987 was exposed to a member of a recently discovered group of S. thermophilus phages (the 987 phage group), generating phage-insensitive mutants, which were then characterized phenotypically and genomically. Decreased phage adsorption was observed in selected bacteriophage-insensitive mutants, and was partnered with a sedimenting phenotype and increased cell chain length or aggregation. Whole genome sequencing of several bacteriophage-insensitive mutants identified mutations located in a gene cluster presumed to be responsible for cell wall polysaccharide production in this strain. Analysis of cell surface-associated glycans by methylation and NMR spectroscopy revealed a complex branched rhamno-polysaccharide in both ST64987 and phage-insensitive mutant BIM3. In addition, a second cell wall-associated polysaccharide of ST64987, composed of hexasaccharide branched repeating units containing galactose and glucose, was absent in the cell wall of mutant BIM3. Genetic complementation of three phage-resistant mutants was shown to restore the carbohydrate and phage resistance profiles of the wild-type strain, establishing the role of this gene cluster in cell wall polysaccharide production and phage adsorption and, thus, infection.     

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.     

Studies on Fungal Tannase

The possibility that selective inhibition of phage by antibiotic may be achieved by using bacterial mutant resistant to the antibiotic was investigated in the system of HM-phages of Clostridium saccharoperbutylacetonicum, a butanol-producing bacterium.

Consequently, it was found that Oxytetracycline, using the antibiotic-resistant mutant as host, inhibited selectively the growth of HM-phages. The bacterial mutant termed type A (one-step mutant resistant to 30 μg/ml of Oxytetracycline) did not permit the growth of HM-phages (HM 2 and HM 3) in the presence of the antibiotic (ca. 10 μg/ml), though it permitted the growth of the phages in the absence of the antibiotic.

An analysis of the mode of action of Oxytetracycline in HM 2-phage system revealed the following, (i) The antibiotic had a slight phagicidal action, (ii) It did not prevent the phage adsorption, (iii) It inhibited the protein synthesis in phage-infected cells, (iv) It inhibited the lysis of infected cells. Active phages were, however, not detected when the lysis-inhibited cells were artificially lysed.

Another type of bacterial mutant was also encountered. In this mutant termed type B the development of resistance to Oxytetracycline (30 μg/ml) was associated with a simultaneous loss of sensitivity to particular phages (HM 2 group).     

Common and form-specific cell wall antigens of Candida albicans as released by chemical and enzymatic treatments

In order to investigate the antigenic properties of the proteins and mannoproteins present in the cell surface of Candida albicans, and to identify individual antigenic moieties and their distribution, a number of polyclonal antisera were obtained by immunizing rabbits with chemical and enzymatic cell wall extracts obtained from intact cells from both growth forms (yeast and mycelium) of the fungus. Prior to injection, wall moieties present in the extracts were subjected to different treatments and/or purification procedures such as adsorption onto polystyrenelatex microbeads or electrophoretic separation. When used as probes in indirect immunofluorescence assays, the different antisera gave rise to different fluorescence patterns varying in intensity and topological localization of the reactivity in C. albicans cells. When the different antisera were used as probes in Western blots of wall proteinaceous materials solubilized from both blastospores and germ tubes, differences in reactivity and specificity were readily discernible, allowing to identify a number of common and form-specific cell wall components. Of special interest was the fact that one of the antisera raised, after adsorption onto heat-killed blastospores, specifically recognized medium to low molecular weight moieties present only in the cell wall extracts from germ tubes. When this antiserum was used as probe in immunofluorescence assays, reactivity was confined to the hyphal extensions. Together, these observations seem to indicate that the different antibody preparations described in this report could represent important tools in the study of different aspects of the cell wall biology in C. albicans, including the identification and study of the distribution of common and form-specific cell wall-bound antigens.Abbreviations IIF Indirect immunofluorescence - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PAb polyclonal antibody