Inhibition Of Sporulation Research Articles

Nucleotide sequences of the Bacillus subtilis flaD locus and a B. licheniformis homologue affecting the autolysin level and flagellation

A DNA fragment containing the flaD locus of Bacillus subtilis, which had been cloned into plasmid pAC3, was subcloned into an M13 phage and sequenced. The sequence contained five open reading frames (ORFs), of which ORF2 was the flaD gene. Unexpectedly, the sequence of the flaD locus was identical to that of sin [sporulation inhibition gene; Gaur, N. K., Dubnau, E. & Smith, I. (1986). Journal of Bacteriology 168, 860-869]. A B. licheniformis homologue (flaL) of the B. subtilis flaD locus was cloned into pUC19 and identified by colony hybridization. The B. licheniformis DNA was subcloned and sequenced. Two ORFs (ORF1, or L-ORF1; and ORF2, or flaL) were detected, encoding 58 and 111 amino acid residues, respectively. These are almost identical in length to ORF1 (D-ORF1; 57 amino acids) and flaD (111 amino acids) on the fragment of B. subtilis DNA. The overall interspecies differences between the nucleotide sequences of D-ORF1 and L-ORF1, and those of flaD and flaL, were 42% and 11%, respectively, and the differences in the predicted amino acid sequences were 50% and 7%, respectively. The regions 3' of the ORFs (flaL and flaD) in both species resemble rho-independent terminators of transcription. The characteristics of the amino acid sequences are also discussed.

Elevated content of hsp 70 does not induce tolerance of sporulation to higher temperature

The temperature permissive for sporulation (up to 42°C) inBacillus megaterium is by 4–5°C lower than that for its growth (up to 46–47°C). The ability ofB. megaterium cells to synthesize and degrade stress proteins under incubation in the sporulation medium was therefore investigated. The higher level of hsp 70, a typical stress protein induced by a temperature shock in postexponential growth phase, did not increase the permissive temperature of sporulation. The hsp 70 protein did not undergo a rapid turnover and its portion in the soluble protein fraction did not drop for at least 6 h at a temperature that was nonpermissive for sporulation (43.5°C). On the other hand, the elevated level of hsp 70 could not bring about the inhibition of sporulation as it was retained in the cells even after a shift of the temperature to 35°C, permitting sporulation of the culture.

A novel Bacillus subtilis gene involved in negative control of sporulation and degradative-enzyme production

We have cloned a 2.5-kilobase fragment of the Bacillus subtilis genomic DNA which caused the reduction of extracellular and cell-associated protease levels when present in high copy number. This fragment, in multicopy, was also responsible for reduced levels of alpha-amylase, levansucrase, alkaline phosphatase, and sporulation inhibition. The gene relevant to this pleiotropic phenotype is referred to as pai. By DNA sequencing, two open reading frames--ORF1 and ORF2, encoding polypeptides of 172 and 207 amino acid residues, respectively--were found. These open reading frames seemed to form an operon. Deletion analysis revealed that an entire region for ORF1 and ORF2 was necessary for the pai phenotype. In addition, it was observed that the presence of the pai gene, in multicopy, caused overproduction of two proteins (molecular masses, 21 and 24 kilodaltons [kDa]). Analyses of the N-terminal amino acid sequences of these two proteins suggested that they were products of ORF1 and ORF2. Disruption of the pai gene at ORF1 in the genomic DNA resulted in the release of repression on protease synthesis and sporulation in glucose-enriched (2%) medium. The mutant carrying insertional disruption at ORF2 could not be constructed, suggesting that the ORF2 product, the 24-kDa protein, is essential for growth. The 21-kDa protein contains a helix-turn-helix domain observed in other DNA-binding proteins. Chromosomal mapping of pai indicated that this gene is located close to thr-5. These results suggest that the pai gene is a novel transcriptional-regulation gene involved in glucose repression.

Sporulation of Botrytis cinerea as affected by photoselective polyethylene sheets and filters

SummarySporulation of Botrytis cinerea was studied in vitro under various filters and polyethylene co‐polymer (PE) sheets. It was found that continuous darkness blocked sporulation completely. Sporulation was inhibited by reduced ultraviolet (u.v.) radiation. Almost total inhibition of sporulation was also found when cultures were grown under a blue filter. The reduction of spore formation was negatively related to the ratio of transmitted blue/u.v. light. The possibility of using this effect to control the incidence of Botrytis cinerea in greenhouses is discussed. A brief report has been published (Reuveni, Raviv, Allingham & Bar, 1988).

Mode of Action of Itraconazole: Morphological Aspects

The broad spectrum of antifungal activity of itraconazole is verified by morphologic criteria at the light and electron microscopical level. Yeast and fungal species examined are Candida albicans, Cryptococcus neoformans, Paracoccidioides brasiliensis, Sporothrix schenckii, Pityrosporum ovale, Trichophyton rubrum and Aspergillus fumigatus. Exposures of cultures of these yeasts and fungi to itraconazole results in dose- and time-dependent alterations which vary in nature and intensity from one species to another. The most striking gross morphological change is seen in the biphasic species and consists of the abolishment of morphogenic development of the blastospore into the hyphal forms (C. albicans and P. ovale) and of the hyphal into the yeast forms (P. brasiliensis). Furthermore, the outgrowth and development of inoculated A. fumigatus hyphae into sporulating vesicles is almost completely abolished. The above mentioned effects on morphogenesis are achieved in the 10(-10)-10(-7) M range. Except for P. ovale, the earliest ultrastructural changes after itraconazole consist of abnormalities at the plasma membrane, the cell wall and cytoplasmic vacuoles. They precede a marked increase in cell volume, defective cell division, abortive hyphal outgrowth and loss of cell viability. These changes are identical to those previously described after miconazole and ketoconazole treatment. However, when compared to the other available azole-derivatives sharing the same basic mechanisms of action, itraconazole displays an exceptionally strong activity against A. fumigatus which can be morphologically translated by a potent necrotizing action on hyphae and inhibition of vesicle formation and sporulation. The in vitro effects of itraconazole are supported by data obtained from microscopic examinations of samples derived from patients with experimental animals infected with various fungal organisms.

Gene dosage effect on the expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium

Significant differences in expression of the δ-endotoxin genes cryA1 and cryA2 of Bacillus thuringiensis subsp. kurstaki were observed in B. subtilis and B. megaterium. The cryA1 gene was expressed when present on a highcopy-number (hen) vector in B. megaterium but not in B. subtilis. The cryA2 gene was expressed in both hosts, but at a higher level in B. megaterium. Expression of the cryA2 gene in B. megaterium was better from a hcn vector than from a low copy number vector. Inhibition of sporulation was observed when the toxin genes were present on hen plasmids in B. subtilis while no such effect was evident in B. megaterium. In addition, there was a significant reduction in copy numbers in both B. subtilis and B. megaterium when δ-endotoxin genes or a spoVG promoter-containing fragment of DNA were cloned into hen plasmids.

Studies on antimicrobial activity of β-hydroxynaphthaldehyde as a possible protectant against biodeterioration

The antimicrobial activity of β-hydroxynaphthaldehyde (HNA) was tested against 40 different fungal species isolated from deteriorated cotton, textiles, canvas, paper, book bindings, photographic films, leather, footwear and other similar articles. The concentrations 10–100 μg/ml were tested by poisoned food technique and 60 μg/ml (0·006%) was found to render 100% inhibition in fungal growth. Further investigations on mode of action were carried out using Aspergillus flavus as a representative fungus. The observations on radial growth revealed a gradual decrease from 13·0 to 4·2 mm at 10·0–50·0 μg/ml concentrations and 28° ± 1° C. Dry weight of mycelium also decreased from 496 to 28·0 mg at the same dosage. After 7 days' incubation no growth was seen at 40 and 50 μg/ml concentrations but, after 12 and 15 days visible growth occured. The effect of HNA on the fungal morphology was loss of colony colour, reduction in head size, shortening and thickening of the mycelium and complete inhibition of sporulation at 50 μg/ml. The results on the secretion of extra-cellular enzymes, viz. amylase, cellulase, protease and lipase, indicated quantitative reduction with the increasing concentrations of HNA. No secretion was found at 40 and 50 μg/ml. This compound was highly effective and possibly behaves as a general cytoplasmic poison. The antimicrobial properties were considered to be due to a highly reactive aldehyde group attached in the β-position in the naphthalene ring. It has been recommended as a long-term protectant against biodeterioration of most of the materials.

Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis.

The hyperproduction of alkaline and neutral proteases is a phenotype of mutation at the hpr locus. This locus has been cloned and sequenced and has been found to code for a protein of 23,718 Mr. The mutations hpr-1, scoC4, and catA7 were identified by sequencing as mutations within the hpr gene. The phenotype of mutations in the hpr gene is due to loss of the hpr gene product, and therefore we suggest that the hpr gene encodes a negative regulator of protease production. This negative regulator must control genes other than protease genes, and these genes must include at least one gene required for sporulation, since overproduction of the hpr gene product by cloning the locus on a multicopy vector results in the inhibition of sporulation as well as protease production. Truncated fragments of the hpr gene or its promoter do not have this phenotype. Transcription of the hpr locus is controlled by the spoOA gene. In an spoOA mutant the hpr gene transcript is constitutively overproduced, as determined by a transcription fusion to beta-galactosidase. The results are consistent with the view that the spoOA gene may control sporulation and transcription by modulating the level and activity of several regulatory proteins.

Co-ordinate control of ammonium-scavenging enzymes in the fruit body cap ofCoprinus cinereusavoids inhibition of sporulation by ammonium

Observation of stained nuclei coupled with determination of activity of NADP-linked glutamate dehydrogenase (NADP-GDH) and glutamine synthetase (GS) in the same tissue, showed that increase in activity of both enzymes was initiated as karyogamy became evident in normally-developing basidia of fruit bodies of the basidiomycete Coprinus cinereus. Derepression of the two enzymes was highly positively correlated (correlation coefficient 0.91), implying co-ordinated regulation. Application and injection of solutions of ammonium salts to Coprinus fruit body caps arrested further development. Other salts had no such effect, indicating a specific sensitivity to ammonium. Similar enzymic events occur during sporulation of Saccharomyces cerevisiae, which is also sensitive to inhibition by ammonium salts. It is concluded that meiosis generally includes ammonium-sensitive processes and that co-ordinated derepression of the ammonium scavenging NADP-GDH and GS is a device to ensure removal of ammonium ions from the meiotic cell.

Co-ordinate control of ammonium-scavenging enzymes in the fruit body cap of Coprinus cinereus avoids inhibition of sporulation by ammonium

Co-ordinate control of ammonium-scavenging enzymes in the fruit body cap of Coprinus cinereus avoids inhibition of sporulation by ammonium

Analysis of the inhibition of sporulation of Bacillus subtilis caused by increasing the number of copies of the spo0F gene.

The Bacillus subtilis spo0F gene was cloned on a 6.3 kbp Bg/II fragment. The effect on sporulation of amplification of the spo0F region was examined. Sporulation was inhibited to less than 5% of that of the parental strain when as few as four copies of the spo0F region were present. Subclones, constructed in autonomous or integrative vectors, were used to demonstrate that the region responsible for the copy-number-dependent asporogeny corresponded closely with the spo0F structural gene. A possible mechanism for this effect is discussed.

Inhibition of sporulation of Phomopsis viticola Sacc., cause of dead arm disease of vines, by fosetyl-Al under field conditions

Several plots of vines, naturally infected with Phomopsis viticola Sacc., were given a single application of fosetyl-Al + folpet at various growth stages starting from stage A in January. At varying intervals after treatment, the spores released from the pycnidia were counted on sections of vine canes in the laboratory. The effectiveness of treatments was also assessed in the field. An inhibition of sporulation of up to 95% occurred, lasting until 2 months after treatment; however, this inhibition, probably caused by fosetyl-Al, can only partly explain the efficacy obtained in the field with these treatments. The fact that the antisporulation activity of fosetyl-Al on P. viticola occurs during the period of vegetative shutdown appears to exclude the involvement of the plant in the mode of action of the fungicide in this case.

Characterization of a cloned Bacillus subtilis gene that inhibits sporulation in multiple copies.

We have isolated a 1.0-kilobase fragment of the Bacillus subtilis chromosome which, when present in high-copy-number plasmids, caused a sporulation-proficient strain to become phenotypically sporulation deficient. This is referred to as the sporulation inhibition (Sin) phenotype. This DNA fragment, in multicopy, also inhibited the production of extracellular protease activity, which normally appears at the beginning of stationary growth. The origin of the fragment was mapped between the dnaE and spo0A genes on the B. subtilis chromosome, and its complete DNA sequence has been determined. By analysis of various deletions and a spontaneous mutant the Sin function was localized to an open reading frame (ORF) predicted from the DNA sequence. Inactivation of this ORF in the chromosome did not affect the ability of cells to sporulate. However, the late-growth-associated production of proteases and alpha-amylase was elevated in these cells. The predicted amino acid sequence of the protein encoded by this ORF had a DNA-binding domain, typically present in several regulatory proteins. We propose that the sin ORF encodes a regulatory protein that is involved in the transition from vegetative growth to sporulation.

Dark reversion of blue-light inhibition of conidiation in Alternaria cichorii

Prolonged exposure to blue light was necessary for almost complete inhibition of sporulation in Alternaria cichorii Nattrass. When a 24-h blue light exposure was followed by darkness, the inhibition of sporulation decreased gradually.

Effect of various growth conditions on spore formation and bacillomycin L production in Bacillus subtilis.

Bacillomycin L is produced by Bacillus subtilis NCIB 8872 in the stationary phase; it is excreted into the culture medium, without prior accumulation in the bacterial cells. The production of bacillomycin L is largely dependent on the composition of the culture medium. The action of specific inhibitors of sporulation, netropsin and diethyl malonate, on antibiotic synthesis is dependent on the composition of the culture medium. Although they occurred at the same time, there appears to be no direct correlation between sporulation and antibiotic synthesis.

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication.

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.

Suppression by temperature of sporulation and of exocellular metalloproteinase synthesis inBacillus megaterium

Temperatures slightly supraoptimal for growth (above 40°C) delayed or inhibited the development of sporangia as well as the synthesis of metalloproteinase. The inhibition of sporulation was reversible and most of the cells sporulated and excreted proteinase when transferred to 35°C. The ability to synthesize the enzyme in a sporulation medium was maximal between 0.5–1.0 h of incubation at 35°C, between 0.5–2.0 h at 43.5°C and even later at 45°C. Proteinase formation was suppressed by temperature at the level of mRNA transcription.

Independent development of heat resistance and ascospores of Hansenula anomala

The development of ascospores and heat resistance was determined in McClary's acetate broth in the presence of two sporulation inhibitors, ethanol and erythromycin. The inhibitors delayed sporulation without preventing development of heat resistance. The presence of ethanol in growth media caused cells to have increased heat resistance. The fatty acid and chitin contents of sporulating Hansenula anomala cells were determined. Highest triglyceride content of cells was associated with the initiation of sporulation. This phenomenon was not observed for phospholipid. As the ratio of saturated to unsaturated fatty acids in triglycerides increased, so did the heat resistance. However, the chitin content of cells was not correlated with heat resistance.

Inhibition of sporulation of Bacillus subtilis by MAPI, a serine protease inhibitor, and interaction of MAPI with membrane bound protease.

At levels of 10^g/ml (≈10_5m), MAPI, a peptidealdehyde type serine protease inhibitor, selectively inhibited sporulation of B. subtilis without appreciably altering exponential growth. The strongest inhibition of sporulation is observed when the inhibitor is added within the first 3~4hr of sporulation.Using [14C]MAPI, the existence of an MAPI-binding substance was investigated. More than 50% of the incorporated MAPI formed a complex with a constituent of the membrane. This substance was identified as the membrane bound protease [Shimizu et al, Agric. Biol. Chem., 47, 1775 (1983)] from chromatographic and enzymatic properties. These results strongly suggested that the inhibition of sporulation of MAPI may be due to repression of proteolytic activity of the membrane bound enzyme through the complex formation. They also suggested that all the essential function of the enzyme was complete before t4.

Sensitivity to ethidium bromide during meiosis in Saccharomyces cerevisiae

Ethidium bromide was found to inhibit nuclear and mitochondrial DNA synthesis during meiosis which resulted in the inhibition of meiotic gene conversion and sporulation and was also lethal. Protection from the effects of ethidium bromide on meiotic gene conversion and survival was found to coincide with DNA synthesis, but it is possible that protection from sporulation inhibition occurs only later in meiosis.