Genetic instability of sporulation-associated characters in a Bacillus subtilis mutant: relationship between sporulation, segregation and the synthesis of extracellular enzymes (kinetic studies).

In the genetically unstable, protease-overproducing 'medusa (M) strains of Bacillus subtilis, segregation of stable, wild-type-like B cells occurred mainly during sporulation. After the end of the exponential growth phase, a small fraction of M cells sporulated quickly and formed M spores, while the majority of the cells, after a 'critical period', gave rise to B segregants which sporulated after a delay. Segregation occurred without cell division. Delayed sporulation, segregation and protease overproduction are related. Similar but more complex results were obtained with the highly unstable TD strains. Sporulation and the kinetics of protease overproduction were also followed in several stable segregants. Depending on the strain, either the rate of protease production or both the rate and time course were affected. The results are interpreted in terms of sequential activation and de-activation of sporulation genes. The production of the alkaline and the neutral proteases was, in general, under common genetic control. In some strains alpha-amylase was also overproduced.     

The isolation and genetic analysis of sporulation-deficient mutants in Saccharomyces cerevisiae

Sporulation-deficient mutants were isolated from a homothallic strain of Saccharomyces cerevisiae. Sporulation was induced in these mutants by procedures to sporulate the products of protoplast fusion between mutants and wild-type strains. Spores formed in this way were crossed to wild-type strains in order to analyze them genetically. Twenty-three genes essential to sporulation were identified by tetrad analysis and complementation tests. Gene symbols spoT1 to spoT23 were tentatively assigned to them. These mutants fell into four classes by examination of premeiotic DNA synthesis and meiotic nuclear division: (i) Premeiotic DNA synthesis did not occur (spoT1 - spoT11); (ii) premeiotic DNA synthesis occurred but meiosis I did not occur (spoT12 - spoT15); (iii) meiosis II did not occur (spoT16 - spoT18); (iv) meiosis II occurred but mature spores were not formed (spoT19 - spoT23). Genes spoT4, spoT8, spoT20, and spoT23 were mapped on chromosomes IV, II, XVI and XI, respectively. SpoT18-1 was a UAG nonsense mutation.     

The diverse sporulation characteristics of Clostridium difficile clinical isolates are not associated with type

Clostridium difficile causes diarrhoeal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis. Endospore production plays a vital role in transmission of infection, and in order to cause disease these spores must then germinate and return to vegetative cell growth. Type BI/NAP1/027 strains of C. difficile have recently become highly represented among clinical isolates and are associated with increased disease severity. It has also been suggested that these 'epidemic' types generally sporulate more prolifically than 'non-epidemic' strains, although the few existing reports are inconclusive and encompass only a small number of isolates. In order to better understand any differences in sporulation rates between epidemic and non-epidemic C. difficile types, we analysed these characteristics using 14 C. difficile clinical isolates of a variety of types. Sporulation rates varied greatly between individual BI/NAP1/027 isolates, but this variation did not appear to be type-associated. Furthermore, a number of BI/NAP1/027 spores appeared to form colonies with a lower frequency than specific non-BI/NAP1/027 strains. The data suggest that (i) careful experimental design is required in order to accurately quantify sporulation; and (ii) current evidence cannot link differences in sporulation rates with the disease severity of the BI/NAP1/027 type.     

Clear, defined medium for the sporulation of Clostridium perfringens.

A new, defined medium for the sporulation of Clostridium perfringens is presented. Sporulation levels exceeding 10(6) to 10(7) heat-resistant spores per ml were obtained for seven strains: PS49, PS52, FD-1, T-65, NCTC strains 8798, 8238, and 10240. In the presence of theophylline, a methylxanthine, higher levels of heat-resistant spores were attained for strains PS49, PS52, FD-1, ant T-65; photomicrographs demonstrated a higher fraction of sporulating cells when these strains were grown in the presence of methylxanthines. Use of washed, highly diluted (less than 100 cells) inocula resulted in no reduction in spore yield. Strain KA3 grew well but sporulated poorly on this medium. The medium was clear and free of precipitate when small amounts (100 microgram/ml) of methylxanthine were incorporated.     

Effect of pH on Sporulation of Bacillus Stearothermophilus

An improved broth medium was developed for high growth yields of Bacillus subtilis var. niger NCIB 8649, Bacillus cereus NCIB 9373, and Bacillus stearothermophilus NCIB 8919 and ATCC 7953. Sporulation was abundant (1.1 times 10-8 B. subtilis var. niger and 9.2 times 10-7 B. cereus per ml) at an initial pH of 7.0. Sporulation of both strains of B. stearothermophilus took place (1.9 times 10-7 and 2.4 times 10-7/ml, respectively) in this medium when initial pH values of 7.7 to 8.7 were used.     

Clear, defined medium for the sporulation of Clostridium perfringens.

A new, defined medium for the sporulation of Clostridium perfringens is presented. Sporulation levels exceeding 10(6) to 10(7) heat-resistant spores per ml were obtained for seven strains: PS49, PS52, FD-1, T-65, NCTC strains 8798, 8238, and 10240. In the presence of theophylline, a methylxanthine, higher levels of heat-resistant spores were attained for strains PS49, PS52, FD-1, ant T-65; photomicrographs demonstrated a higher fraction of sporulating cells when these strains were grown in the presence of methylxanthines. Use of washed, highly diluted (less than 100 cells) inocula resulted in no reduction in spore yield. Strain KA3 grew well but sporulated poorly on this medium. The medium was clear and free of precipitate when small amounts (100 microgram/ml) of methylxanthine were incorporated.     

Genomic diversity of cultivable Lactobacillus populations residing in the neonatal and adult gastrointestinal tract

The objective of this study was to investigate the cultivable Lactobacillus population in adult and infant faecal material to identify strains shared across a number of individuals. A range of lactobacilli isolated on Lactobacillus-selective agar from faeces of 16 infants and 11 adults were genetically fingerprinted and further characterized by 16S rRNA gene sequencing. The relatedness of all the Lactobacillus strains isolated to known species was also determined both genetically and phenotypically. This study revealed that the human intestine is initially colonized by only a few (1-2) different cultivable strains whereas in adults the pattern becomes more complex with a higher diversity of strains. The adult samples contained three genetically distinct Lactobacillus strains in some cases, while infant samples generally harboured only one dominant Lactobacillus strain. Moreover, the species in general appeared to differ with Lactobacillus rhamnosus and Lactobacillus casei/paracasei found mainly in adults, whereas Lactobacillus gasseri and Lactobacillus salivarius were more commonly isolated in infant samples. The data reaffirm the differences in Lactobacillus populations both between individual subjects and between the infant and adult, with an overall change in the diversity and complexity from early stages of life to adulthood.     

Genetic control of nucleolar number in mouse

The number of nucleoli per cell was studied in embryonic tissues and neonatal livers of the mouse. Different inbred strains showed different mean nucleolar numbers. The f₁, F₂, and backcross progeny between two inbred strains, BALB/c and C3H, revealed that the number of nucleoli per cell was genetically determined and probably follows polygenic inheritance.     

Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed. A minimum of ca. 4 cells per microbead was necessary for efficient lysis and sporulation to proceed. Increasing this number accelerated the lysis and sporulation process. No lysis occurred when an average of 0.4 cell was entrapped per bead. Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells. Sporulation and lysis also depended upon the cell density in the culture as a whole. The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M. xanthus.     

Sporulation Synchrony of Saccharomyces cerevisiae Grown in Various Carbon Sources

Sporulation of several strains of Saccharomyces cerevisiae grown in a variety of carbon sources that do not repress the tricarboxylic acid cycle enzymes was more synchronous than the sporulation of cells grown in medium containing dextrose which does repress those enzymes. Dextrose-grown cells showed optimal sporulation synchrony when inoculated into sporulation medium from early stationary phase when the dextrose in the medium is exhausted. Logarithmic-phase cells grown in either non-fermentable carbon sources (acetate and glycerol) or a fermentable carbon source that does not repress tricarboxylic acid cycle enzymes (galactose) sporulated more synchronously than the early stationary-phase dextrose cells. Attempts were made to sporulate cells taken from both complex and semidefined media. The semidefined acetate medium failed to support the growth of a number of strains. However, cells grown in the complex acetate medium, as well as both complex and semidefined glycerol and galactose media, sporulated with better synchrony than did the dextrose-grown cells.     

Genetic diversity in strains of the genus Anabaena isolated from planktonic and benthic habitats of the Gulf of Finland (Baltic Sea)

Late summer cyanobacterial blooms in the Baltic Sea contain Anabaena sp. together with Nodularia spumigena and Aphanizomenon flos-aquae. Although Anabaena is common especially in the Gulf of Finland, very little is known about its genetic diversity. Here we undertook a molecular phylogenetic study of 68 Anabaena strains isolated from the brackish Gulf of Finland. We sequenced the 16S rRNA genes from 54 planktonic and 14 benthic Anabaena strains, and rbcL and rpoC1 genes from a subset of these strains. Phylogenetic trees showed that Anabaena strains, from both planktonic and benthic habitats, were genetically diverse. Although the Anabaena strains were morphologically diverse, in our study only one genetically valid species was found to exist in the plankton. Evolutionary distances between benthic Anabaena strains were greater than between planktonic strains, suggesting that benthic habitats allow for the maintenance of greater genetic diversity than planktonic habitats. A number of novel lineages containing only sequences obtained in this study were compiled in the phylogenetical analyses. Thus, it seemed that novel lineages of the genus Anabaena may be present in the Baltic Sea. Our results demonstrate that the Baltic Sea Anabaena strains show surprisingly high genetic diversity.     

Catabolite repression vs derepression, an approach to differentiation during sporulation in Bacillus subtilis

Glutamine, like glucose, repressed sporulation and the synthesis of mycobacillin and dipicolinic acid by Bacillus subtilis , and these syntheses were derepressed by dibutyryl cyclic GMP but not by dibutyryl cyclic AMP. Neither of these dibutyryl cyclic nucleotides affected sporulation or a number of spore-associated parameters in the strain under normal physiological conditions. Mutants insensitive to glutamine repression were indifferent to the addition of either of the dibutyryl cyclic nucleotides both in the presence and in the absence of glutamine. Sporulation resulted from the remission of repression obtained under the catabolically active state.     

Diallel genetic analysis of the elements of paradise fish's (Macropodus opercularis L.) behavior in familiar and novel situations

Elements of the paradise fish's ethogram were recorded in 1 familiar and 3 different unfamiliar situations and the inheritance of these behavioral elements was investigated employing a five times replicated diallel cross between 3 inbred strains. A generalized Hayman Analysis of Variance and a Variance Covariance Analysis were performed to estimate genetic effects and parameters, such as, additive genetic variance, different sorts of dominance variance, reciprocal effects, direction and degree of dominance, ratio between the frequency of dominant and of recessive alleles, minimum number of effective factors and heritabilities, etc. Knowing the genetic architecture, we make inferences about the possible evolutionary past of the behavioral elements and explain why selection might favor certain types of paradise fish's behavior in particular circumstances. In several cases a possibility of "monogenic" inheritance emerged. We explain this finding and conclude that in a cross experiment where the inheritance of phenotypical units are investigated by employing only a few genetically different strains this result may be expected.     

SAD mutation of Saccharomyces cerevisiae is an extra a cassette.

Sporulation of Saccharomyces cerevisiae ordinarily requires the a1 function of the a mating type locus. SAD is a dominant mutation that allows strains lacking a1 (MAT alpha/MAT alpha and mata1/MAT alpha diploids) to sporulate. We provide functional and physical evidence that SAD is an extra cassette in the yeast genome, distinct from those at HML, MAT, and HMR. The properties of SAD strains indicate that the a cassette at SAD produces a limited amount of a1 product, sufficient for promoting sporulation but not for inhibiting mating and other processes. These conclusions come from the following observations. (i) SAD did not act by allowing expression of HMRa: mata1/MAT alpha diploids carrying SAD and only alpha cassettes at HML and HMR sporulated efficiently. (ii) SAD acted as an a cassette donor in HML alpha HMR alpha strains and could heal a mata1 mutation to MATa as a result of mating type interconversion. (iii) The genome of SAD strains contained a single new cassette locus, as determined by Southern hybridization. (iv) Expression of a functions from the SAD a cassette was limited by Sir: sir- SAD strains exhibited more extreme phenotypes than SIR SAD strains. This observation indicates that SAD contains not only cassette information coding for a1 (presumably from HMRa) but also sites for Sir action.     

Studies on lipopeptide biosynthesis by Bacillus subtilis: Isolation and characterization of iturin−, surfactin+ mutants

Abstract Two mutant strains, M35 and M89, were obtained by UV irradiation from a wild-type Bacillus subtilis producing iturin and surfactin. Sporulation and surfactin production were similar in both mutants and in the parent strain, while the iturin production of M35 was 300-fold less than that of the wild-type strain; M89 did not produce any iturin. The analysis of the incorporation of sodium [1-¹⁴C]acetate into cellular lipids and lipopeptides showed that M89 still synthesized β-amino fatty acids, the lipid moiety of iturin.     

Catabolite repression vs derepression, an approach to differentiation during sporulation in Bacillus subtilis.

Glutamine, like glucose, repressed sporulation and the synthesis of mycobacillin and dipicolinic acid by Bacillus subtilis, and these syntheses were depressed by dibutyryl cyclic GMP but not by dibutyryl cyclic AMP. Neither of these dibutyryl cyclic nucleotides affected sporulation or a number of spore-associated parameters in the strain under normal physiological conditions. Mutants insensitive to glutamine repression were indifferent to the addition of either of the dibutyryl cyclic nucleotides both in the presence and in the absence of glutamine. Sporulation resulted from the remission of repression obtained under the catabolically active state.     

Sporulation Genes Associated with Sporulation Efficiency in Natural Isolates of Yeast

Yeast sporulation efficiency is a quantitative trait and is known to vary among experimental populations and natural isolates. Some studies have uncovered the genetic basis of this variation and have identified the role of sporulation genes (IME1, RME1) and sporulation-associated genes (FKH2, PMS1, RAS2, RSF1, SWS2), as well as non-sporulation pathway genes (MKT1, TAO3) in maintaining this variation. However, these studies have been done mostly in experimental populations. Sporulation is a response to nutrient deprivation. Unlike laboratory strains, natural isolates have likely undergone multiple selections for quick adaptation to varying nutrient conditions. As a result, sporulation efficiency in natural isolates may have different genetic factors contributing to phenotypic variation. Using Saccharomyces cerevisiae strains in the genetically and environmentally diverse SGRP collection, we have identified genetic loci associated with sporulation efficiency variation in a set of sporulation and sporulation-associated genes. Using two independent methods for association mapping and correcting for population structure biases, our analysis identified two linked clusters containing 4 non-synonymous mutations in genes – HOS4, MCK1, SET3, and SPO74. Five regulatory polymorphisms in five genes such as MLS1 and CDC10 were also identified as putative candidates. Our results provide candidate genes contributing to phenotypic variation in the sporulation efficiency of natural isolates of yeast.     

Effects of major spore-specific DNA binding proteins on Bacillus subtilis sporulation and spore properties

Sporulation of a Bacillus subtilis strain (termed alpha(-) beta(-)) lacking the majority of the alpha/beta-type small, acid-soluble spore proteins (SASP) that are synthesized in the developing forespore and saturate spore DNA exhibited a number of differences from that of the wild-type strain, including delayed forespore accumulation of dipicolinic acid, overexpression of forespore-specific genes, and delayed expression of at least one mother cell-specific gene turned on late in sporulation, although genes turned on earlier in the mother cell were expressed normally in alpha(-) beta(-) strains. The sporulation defects in alpha(-) beta(-) strains were corrected by synthesis of chromosome-saturating levels of either of two wild-type, alpha/beta-type SASP but not by a mutant SASP that binds DNA poorly. Spores from alpha(-) beta(-) strains also exhibited less glutaraldehyde resistance and slower outgrowth than did wild-type spores, but at least some of these defects in alpha(-) beta(-) spores were abolished by the synthesis of normal levels of alpha/beta-type SASP. These results indicate that alpha/beta-type SASP may well have global effects on gene expression during sporulation and spore outgrowth.     

Sporulation in Bacillus licheniformis during altered bacitracin synthesis

Sporulation in different strains of Bacillus licheniformis, 10716 and 1001 in connection with changes in synthesis of bacitracin was studied. It was shown that the sporulation efficiency did not depend on the synthesis of the antibiotic: in some strains with low potency for the antibiotic production, the sporulation level was lowered, while in the others, it was not lowered. Moreover, normal sporulation was also observed, when the synthesis of bacitracin was inhibited. Therefore, it is suggested that there is no correlation between the sporulation and antibiotic production.     

Salinity tolerance of Saprolegnia parasitica coker

Experiments on two strains ofS. parasitica have shown that this species together with other species ofSaprolegnia have a high tolerance to salinity when vegetative growth alone is considered. The effect of temperature and salinity on asexual sporulation shows that at temperatures of up to 20°C good sporulation occurs in salinities of 3.5%.. At temperatures above this, sporulation is repressed and gammae tend to be formed. Temperature appears to have a marked effect on the initial germination of zoospores, but after 12 hours this effect is minimized.

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Zusammenfassung Untersuchungen an zwei Stämmen vonS. parasitica haben gezeigt, daß diese Art, zusammen mit anderen Arten vonSaprolegnia, eine hohe Toleranz gegen Salinität hat, wenn das vegetative Wachstum allein in Betracht gezogen wird. Die Wirkung von Temperatur und Salinität an asexuellen Sporulation zeigt, daß bei Temperaturen bis zu 20°C eine gute Sporulation in einer Salinität von 3.5%. vorherrscht. Bei höheren Temperaturen wird Sporulation unterdrückt und bilden sich Gammae. Temperatur scheint eine bedeutende Wirkung an der initialen Germination von Zoosporen zu haben, jedoch ist diese Wirkung nach 12 Stunden minimal.