Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe Streptomyces coelicolor A3(2)

The Gram‐positive aerobe Streptomyces coelicolor undergoes a complex life cycle including growth as vegetative hyphae and the production of aerial hyphae and spores. Little is known about how spores retain viability in the presence of oxygen; however, nothing is known about this process during anaerobiosis. Here, we demonstrate that one of the three respiratory nitrate reductases, Nar‐1, synthesized by S. coelicolor is functional exclusively in spores. A tight coupling between nitrite production and the activity of the cytoplasmically oriented Nar‐1 enzyme was demonstrated. No exogenous electron donor was required to drive nitrate reduction, which indicates that spore storage compounds are used as electron donors. Oxygen reversibly inhibited nitrate reduction by spores but not by spore extracts, suggesting that nitrate transport might be the target of oxygen inhibition. Nar‐1 activity required no de novo protein synthesis indicating that Nar‐1 is synthesized during sporulation and remains in a latently active state throughout the lifetime of the spore. Remarkably, the rates of oxygen and of nitrate reduction by wetted spores were comparable. Together, these findings suggest that S. coelicolor spores have the potential to maintain a membrane potential using nitrate as an alternative electron acceptor.     

Biotransformation of antibiotics

Summary The absence of a lag period in the bioconversion of chloramphenicol by spores and the pronounced influence of the pH of the medium on this reaction strongly suggested that chloramphenicol acetyltransferase (CAT) is located at or near the surface of the spores of Streptomyces griseus. A two-hour exposure of spores to dilute solutions of -mercaptoethanol or surfactants resulted in significant decrease in activity even in the presence of glucose as an energy source. However, the inclusion of any of the reagents in the reaction mixture neither influenced the conversion activity nor the spore viability. These treatments did not reveal any cryptic activity for CAT in the spores. In addition, more drastic treatment of the spores with ethylenediaminetetraacetate (EDTA) or with concentrated salt solutions did not reduce the activity nor significantly affect the spore viability. Considering the modes of action of -mercaptoethanol and the surfactants, a combination of disulfide bridges and lipoprotein interactions may be responsible for the binding of CAT to the surface of the spore. Moreover, results of acid treatment of intact spores indicated that most of CAT activity, if not all, is located at the spore surface. Incorporation of ¹⁴C-acetate by cell-free extracts of Streptomyces griseus clearly showed that CAT selectively catalyzed the formation of chloramphenicol-3-acetate at an optimum pH of 6.5. The shape of the pH-activity curve in cell-free extracts is essentially identical to that of the enzyme in intact spores and is additional evidence that the enzyme is located at the spore surface.     

Bacterial Surface Display of a Co-Factor Containing Enzyme, ω-Transaminase from Vibrio fluvialis Using the Bacillus subtilis Spore Display System

To improve the conventional bacterial surface display systems and to display a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis, which needs pyridoxal phosphate (PLP) for efficient transamination, Bacillus subtilis spore display system with cotG, as an anchoring motif was used. Flow cytometry of the B. subtilis spore-expressing ω-transaminase proved its surface localization on the spore. The enzymatic activity of the spore expressing ω-transaminase was more than 30 times higher than that of the host spore. Protease treatment of the ω-transaminase displaying spores resulted in decreased transaminase activity, which is in keeping with the surface location of the fusion protein, CotG-ω-transaminase.     

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Hydrolytic enzyme activities in germinating spores ofAdiantum capillus-veneris L.

Changes in hydrolytic enzyme activities were investigated during spore germination ofAdiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imbibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination.     

Purification and properties of a germination-specific cortex-lytic enzyme from spores of Bacillus megaterium KM.

Two peptidoglycan-lytic enzyme activities were isolated from spores of Bacillus megaterium KM. Surface-bound lytic enzyme was extracted from dormant spores and hydrolysed a variety of peptidoglycan substrates including isolated spore cortex, but did not cause refractility changes in permeabilized spores. Germination-specific lytic enzyme activity appeared early in germination and had minimal activity on isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus isolated peptidoglycan substrates, but caused refractility changes in permeabilized spores of several Bacillus species. The germination-specific lytic enzyme was shown to be a heat-sensitive 29 kDa protein with maximal activity at pH 6.5. It catalysed post-commitment muramic acid delta-lactam synthesis and displayed an inhibitor profile similar to that for post-commitment A600 loss. The relationship of the germination-specific enzyme to a recently proposed model of spore germination is discussed.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Fine Structure of Five Species of Myxomycetes with Clustered Spores*

SYNOPSIS. Electronmicroscopic studies were made of 5 species of Myxomycetes with clustered spores to determine the exact mechanism holding the spores together. In Dianema corticatum the neighboring spore walls were closely appressed and appeared to be fused at numerous points. In Trichia synsporum there was a substance between the outer spore walls of adjacent spores which seemed to cement them together. In Badhamia nitens the spore walls were completely fused along their closely appressed surfaces. The adjacent spores of Badhamia versicolor appeared to be free, or if any fusion point was present it was only at the tips of the ornamentation. In Physarum bogoriense such tips were in contact with those of neighboring spores and were occasionally fused at these points.     

Mitochondrial biogenesis during fungal spore germination. Development of cytochrome c oxidase activity.

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-l-cysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80-incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de novo upon germination.     

A MORPHOMETRIC ANALYSIS OF CYTOLOGICAL CHANGES DURING SPORE MATURATION IN THE MYXOMYCETE DIDYMIUM IRIDIS

Ultrastructure of spore maturation in the myxomycete Didymium iridis was investigated using morphometric analytical techniques. Changes in actual volume (μm³) and relative volume (Vv) of nuclei, autophagic vacuoles, mitochondria, microbodies, lipid droplets, and spore wall were described for spores in three stages of development. Stage I spores were newly formed, surrounded only by the cell membrane. Stage II spores were approximately 1 hr older than Stage I spores and possessed surface spines, but little if any additional wall material. Stage III spores were 24 hr old and possessed a fully formed, multilayered wall. The results of this study indicate that spore maturation in D. iridis is a multistep process involving a decrease in spore volume and coordinated changes in specific organelle compartments. From Stage I to Stage III, mean spore volume decreased by more than 50%. Percent volume data (Vv) showed that Stage I spores allocated volume equally to all measured organelles except microbodies and the spore wall, the latter of which had not yet begun to develop. By Stage II, only the nucleus and spore wall showed significant changes in Vv values, both increasing. In terms of actual volume, the nucleus, autophagic vacuole and spore wall increased by Stage II. Between Stages II and III the cell wall was the only component to increase in volume, all others decreased in volume. Our data indicate a close relationship between a decrease in organelle volume and an increase in cell wall volume in the Stage III spore. The autophagic vacuole and the cell wall dominated the volume of the Stage III spore while the remaining volume was allocated unequally to the other components.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.     

Intradiurnal periodicity of fungal spore concentrations (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) in Cracow, Poland

Aerobiological monitoring enables the definition of seasonal fungal spore concentrations and also intradiurnal time when the highest concentrations of spores could cause or increase allergy symptoms. These data are useful to estimate symptoms of disease, duration of infection and how advanced the illness is in people suffering from fungal allergens. The aim of the study was to compare the concentrations of fungal spores (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) during dry and rainy periods and to analyse their intradiurnal changes. Average daily spore concentrations in dry and rainy periods were compared, using z test, separately for each taxon, season and for a combined 3-year period. Intradiurnal periodicity of fungal spore concentrations was analysed on the basis of three complementary diagrams. These spore concentrations were presented using three curves for all, dry and rainy days in 1997–1999 (April–November). The spore percentage in particular hours was normalized in relation to the daily spore sum accepted as 100%. Two further diagrams enabled the more precise analysis of the highest concentrations in dry days. Daily Botrytis and Cladosporium spore concentrations did not show significant differences between dry and rainy periods. In the case of Didymella and Ganoderma spore concentrations, there were no significant differences between both weather types in the single years, although there was a significant difference when a 3-year period was considered. The differences between daily concentrations of Alternaria spores in dry and rainy periods occurred in 1997 and in a 3-year period. Intradiurnal periodicity of spore concentrations was different for ‘dry’ and ‘wet’ fungal spores. Dry spores are released from the spore-producing parts of the fungus under conditions of decreasing humidity and increasing airflow. Examples of dry spores are those from Alternaria, Cladosporium and Botrytis. Wet spores, such as those from many Ascomycetes (Didymella) and Basidiomycetes (Ganoderma), are released into the atmosphere by processes related to humidity conditions or rain. The highest concentrations of ‘dry’ spores were observed early in the afternoon, while highest values of ‘wet’ spore concentrations occurred in the predawn hours. Statistically non-significant differences between daily spore concentrations in dry and rainy periods of single seasons were found except for Alternaria. Statistically significant differences could occur when the studied period was longer than one season (Alternaria, Didymella, Ganoderma). The highest concentrations of Alternaria, Botrytis and Cladosporium spores were recorded at noon and early in the afternoon. Concentrations of Didymella and Ganoderma spores were highest in the predawn hours.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra

Aims: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. Materials and Results: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair‐like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH‐sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore’s negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. Conclusions: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. Significance and Impact of the Study: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.     

Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis

The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca(2+)-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.     

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883. © 2010 Wiley Periodicals, Inc.     

Water potential,glycerol synthesis,and water content of germinating Phycomyces spores

During early germination, the sporangiospores of Phycomyces blakesleeanus synthesized large amounts of glycerol. Glycerol started leaking out of the spores after some 20 min germination. Simultaneously the water content of the spores greatly increased. Water uptake was accompanied by disapperance of the phase contrast halo and an increase in spore cross-sectional area which all occurred during the same period between 10 and 30 min germination. When spores were incubated in 0.5 or 1 M sucrose, glycerol accumulated in the spores to much higher concentrations and the increase in cellular water content was greatly reduced and retarded. Glycerol synthesis and the concomitant lowering of spore osmotic potential was not the only mediator of spore swelling since equally important glycerol concentrations loaded into dormant spores did not cause spore water uptake or swelling. Also the swelling of the spores was less affected than water uptake by decreases in ambient water potential. Apparently also cell wall loosening was involved in the swelling phenomenon which might have important implications for cellular metabolism.     

Airborne fungal spores in a sawmill environment in Palakkad District, Kerala, India

Concentration of airborne fungal spores inindoor and outdoor environments of a sawmill in Palakkad district of Kerala, India was studied with Burkard Personal Slide Sampler from January to December 1997. Total spore concentration in the indoor and outdoor showed a 3:2 ratio. Higher spore count was observed in indoor in January and in outdoor in October. Thirty three fungal spore types were identified from the indoor and twenty six from the outdoor. Aspergillus/Penicillium, Cladosporium, Nigrospora, Ganoderma, `other basidiospores' and ascospores were the dominant components of the airspora. Aspergillus/Penicillium, the most dominant spore type in the indoor contributed 51.19% and Cladosporium, the most dominant spore type in the outdoor contributed 44.75% of the total spores. The study revealed high prevalence of predominantly allergenic fungal spores in the sawmill environment.