Levels of glycine betaine in growing cells and spores of Bacillus species and lack of effect of glycine betaine on dormant spore resistance

Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased approximately 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and > or =20-fold lower in B. cereus and B. megaterium spores. Although GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.     

Roles of DacB and spm proteins in clostridium perfringens spore resistance to moist heat, chemicals, and UV radiation

Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that typically possess high spore heat resistance. Previous studies have shown that alpha/beta-type small, acid-soluble proteins (SASP) play a major role in the resistance of Bacillus subtilis and C. perfringens spores to moist heat, UV radiation, and some chemicals. Additional major factors in B. subtilis spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the spm and dacB gene products and the sporulation temperature. In the current work, we have shown that the spm and dacB genes are expressed only during C. perfringens sporulation and have examined the effects of spm and dacB mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation, and a number of chemicals. The results of these analyses indicate that for C. perfringens SM101 (i) core water content and, probably, cortex PG structure have little if any role in spore resistance to UV and formaldehyde, presumably because these spores' DNA is saturated with alpha/beta-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and, probably, cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures, resulting in spores that are more resistant to moist heat; and (v) factors in addition to SpmAB, DacB, and sporulation temperature play roles in determining spore core water content and thus, spore resistance to moist heat.     

Difference between the spore sizes of Bacillus anthracis and other Bacillus species

AIMS: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0.81 +/- 0.08 microm and 0.86 +/- 0.08 microm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1.26 +/- 0.13 microm or shorter, and another group of strains with mean spore lengths between 1.49 and 1.67 microm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. CONCLUSIONS: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.     

A procedure for high-yield spore production by Bacillus subtilis

Bacillus subtilis spores have a number of potential applications, which include their use as probiotics and competitive exclusion agents to control zoonotic pathogens in animal production. The effect of cultivation conditions on Bacillus subtilis growth and sporulation was investigated in batch bioreactions performed at a 2-L scale. Studies of the cultivation conditions (pH, dissolved oxygen concentration, and media composition) led to an increase of the maximum concentration of vegetative cell from 2.6 x 10(9) to 2.2 x 10(10) cells mL(-)(1) and the spore concentration from 4.2 x 10(8) to 5.6 x 10(9) spores mL(-)(1). A fed-batch bioprocess was developed with the addition of a nutrient feeding solution using an exponential feeding profile obtained from the mass balance equations. Using the developed feeding profile, starting at the middle of the exponential growth phase and finishing in the late exponential phase, an increase of the maximum vegetative cell concentration and spore concentration up to 3.6 x 10(10) cells mL(-)(1) and 7.4 x 10(9) spores mL(-)(1), respectively, was obtained. Using the developed fed-batch bioreaction a 14-fold increase in the concentration of the vegetative cells was achieved. Moreover, the efficiency of sporulation under fed-batch bioreaction was 21%, which permitted a 19-fold increase in the final spore concentration, to a final value of 7.4 x 10(9) spores mL(-)(1). This represents a 3-fold increase relative to the highest reported value for Bacillus subtilis spore production.     

Cortex content of asporogenous mutants of Bacillus subtilis.

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none.     

Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores.

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.     

Cloning, nucleotide sequencing, and genetic mapping of the gene for small, acid-soluble spore protein gamma of Bacillus subtilis.

The Bacillus subtilis gene (sspE) which codes for small acid-soluble spore protein gamma (SASP-gamma) was cloned, and its chromosomal location (65 degrees, linked to glpD) and nucleotide sequence were determined. The amino acid sequence of SASP-gamma is similar to that of SASP-B of Bacillus megaterium, but these sequences are not as highly conserved across species as are those of other SASPs. The SASP-gamma gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is approximately 340 nucleotides long. The results of hybridization of an sspE gene probe to Southern blots of B. subtilis DNA suggested that there is only a single gene coding for the SASP-gamma type of protein in B. subtilis. This was confirmed by introducing a deletion mutation into the cloned sspE gene and transferring the deletion into the B. subtilis chromosome, with concomitant loss of the wild-type gene. This sspE deletion strain sporulated well, but lacked the SASP-gamma type of protein.     

苏云金芽孢杆菌gerA操纵子在芽孢萌发中的作用

芽孢萌发的营养诱导剂通过与特异的萌发受体结合激活下游的萌发过程,从而使芽孢经过一系列的遗传变化及生化反应恢复营养生长.从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆到一个与枯草芽孢杆菌(Bacillus subtilis)gerA操纵子和蜡状芽孢杆菌(Bacillus cereus)gerR操纵子同源的gerA操纵子.苏云金芽孢杆菌gerA操纵子含有3个开放读码框:gerAA、gerAC和gerAB,该操纵子在产孢起始3个小时后开始转录.gerA的破坏阻断了L-丙氨酸诱导的芽孢萌发并且延迟了肌苷诱导的萌发.在L-丙氨酸诱导芽孢萌发的过程中D-环丝氨酸能够提高芽孢的萌发率.     

Sporulation in Bacillus subtilis. Antigenic changes during spore formation

1. Antisera, prepared against extracts of cells and spores of Bacillus subtilis, were used in immunoelectrophoretic studies of the changes occurring in cell extracts during the course of spore formation. 2. At least 15 antigens could be detected in vegetative-cell extracts by the antiserum prepared against cell extracts and at least seven could be demonstrated in spore extracts by the homologous antiserum. 3. Cross-absorption studies showed that two of these antigens were probably completely specific for vegetative-cell extracts and that one was probably completely specific for spore extracts. The remainder were probably present in very small quantities in the heterologous extract. 4. In extracts of cells sporulating in an ;exhaustion medium' those antigens characteristic of the spore began to appear about 1hr. after the end of exponential growth. 5. In cells sporulating in a resuspension medium, spore antigens were detected at 4hr., and by 7hr. a decrease in vegetative-cell antigens was observed. 6. In an asporogenous mutant blocked early in sporulation there was neither an increase in spore antigens nor a decrease in vegetative-cell antigens. 7. In an asporogenous mutant blocked later in sporulation, there was an increase in spore antigens similar to that which occurred in the sporogenous strain.     

From fundamental studies of sporulation to applied spore research

Sporulation in the Gram-positive bacterium, Bacillus subtilis, has been used as an excellent model system to study cell differentiation for almost half a century. This research has given us a detailed picture of the genetic, physiological and biochemical mechanisms that allow bacteria to survive harsh environmental conditions by forming highly robust spores. Although many basic aspects of this process are now understood in great detail, including the crystal and NMR structures of some of the key proteins and their complexes, bacterial sporulation still continues to be a highly attractive model for studying various cell processes at a molecular level. There are several reasons for such scientific interest. First, some of the complex steps in sporulation are not fully understood and/or are only described by 'controversial' models. Second, intensive research on unicellular development of a single microorganism, B. subtilis, left us largely unaware of the multitude of diverse sporulation mechanisms in many other Gram-positive endospore and exospore formers. This diversity would likely be increased if we were to include sporulation processes in the Gram-negative spore formers. Spore formers have great potential in applied research. They have been used for many years as biodosimeters and as natural insecticides, exploited in the industrial production of enzymes, antibiotics, used as probiotics and, more, exploited as possible vectors for drug delivery, vaccine antigens and other immunomodulating molecules. This report describes these and other aspects of current fundamental and applied spore research that were presented at European Spores Conference held in Smolenice Castle, Slovakia, June 2004.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Effects of phosphorelay perturbations on architecture, sporulation, and spore resistance in biofilms of Bacillus subtilis

The spore-forming bacterium Bacillus subtilis is able to form highly organized multicellular communities called biofilms. This coordinated bacterial behavior is often lost in domesticated or laboratory strains as a result of planktonic growth in rich media for many generations. However, we show here that the laboratory strain B. subtilis 168 is still capable of forming spatially organized multicellular communities on minimal medium agar plates, exemplified by colonies with vein-like structures formed by elevated bundles of cells. In line with the current model for biofilm formation, we demonstrate that overproduction of the phosphorelay components KinA and Spo0A stimulates bundle formation, while overproduction of the transition state regulators AbrB and SinR leads to repression of formation of elevated bundles. Time-lapse fluorescence microscopy studies of B. subtilis green fluorescent protein reporter strains show that bundles are preferential sites for spore formation and that flat structures surrounding the bundles contain vegetative cells. The elevated bundle structures are formed prior to sporulation, in agreement with a genetic developmental program in which these processes are sequentially activated. Perturbations of the phosphorelay by disruption and overexpression of genes that lead to an increased tendency to sporulate result in the segregation of sporulation mutations and decreased heat resistance of spores in biofilms. These results stress the importance of a balanced control of the phosphorelay for biofilm and spore development.     

Altered arrangement of proteins in the spore coat of a germination mutant of Bacillus subtilis

Spores produced by a mutant of Bacillus subtilis were slow to develop their resistance properties during sporulation, and were slower to germinate than were wild-type spores. The coat protein composition of the mutant spores, as analysed by SDS-PAGE, was similar to that of the wild-type spores. However, one of the proteins (mol. wt 12000) which is normally present in the outer-most layers of mature wild-type spores and which is surface-exposed, was assembled abnormally into the coat of the mutant spores and not surface-exposed. The mutation responsible for this phenotype (spo-520) has been mapped between pheA and leuB on the B. subtilis chromosome, and was 47% cotransformable with leuB16. This mutation, and three others closely linked to it, define a new sporulation locus, spoVIB, which is involved in spore coat assembly. The phenotype of the mutant(s) supports the contention that spore germination and resistance properties may be determined by the assembly of the coat.     

Mechanisms of Bacillus subtilis spore killing by and resistance to an acidic Fe-EDTA-iodide-ethanol formulation

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to an acidic solution containing Fe(3+), EDTA, KI and ethanol termed the KMT reagent. METHODS AND RESULTS: Wild-type B. subtilis spores were not mutagenized by the KMT reagent but the wild-type and recA spores were killed at the same rate. Spores (alpha(-)beta(-)) lacking most DNA-protective alpha/beta-type small, acid-soluble spore proteins were less resistant to the KMT reagent than wild-type spores but were also not mutagenized, and alpha(-)beta(-) and alpha(-)beta(-)recA spores exhibited nearly identical resistance. Spore resistance to the KMT reagent was greatly decreased if spores had defective coats. However, the level of unsaturated fatty acids in the inner membrane did not determine spore sensitivity to the KMT reagent. Survivors in spore populations killed by the KMT reagent were sensitized to killing by wet heat or nitrous acid and to high salt in plating medium. KMT reagent-killed spores had not released their dipicolinic acid (DPA), although these killed spores released their DPA more readily when germinated with dodecylamine than did untreated spores. However, KMT reagent-killed spores did not germinate with nutrients or Ca(2+)-DPA and were recovered only poorly by lysozyme treatment in a hypertonic medium. CONCLUSIONS: The KMT reagent does not kill spores by DNA damage and a major factor in spore resistance to this reagent is the spore coat. KMT reagent treatment damages the spore's ability to germinate, perhaps by damaging the spore's inner membrane. However, this damage is not oxidation of unsaturated fatty acids. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanism of spore resistance to and killing by the KMT reagent developed for killing Bacillus spores.     

Analysis of damage due to moist heat treatment of spores of Bacillus subtilis

Aims:  To determine conditions for generation and recovery of Bacillus subtilis spore populations heavily damaged by moist heat treatment.
Methods and Results:  Bacillus subtilis spores were treated with moist heat and spore viability was assessed on different media. A rich medium and several minimal media gave similar spore recoveries after moist heat treatment, but lack of glucose in minimal media greatly decreased spore recovery. High NaCl levels also greatly decreased the recovery of moist heat-treated spores on minimal media, and addition of good osmoprotectants reversed this effect. Moist heat treatment did not decrease spore recovery on minimal media with high salt through DNA damage or by eliminating spore germination, but by affecting spore outgrowth.
Conclusions:  Conditions for generating B. subtilis spore populations with high levels of conditional moist heat damage have been determined. The major conditional damage appears to be in spore outgrowth, perhaps because of damage to one or more important metabolic enzymes.
Significance and Impact of the Study:  This work has provided new insight into the mechanism of B. subtilis spore killing by moist heat.     

Resporulation of outgrowing Bacillus subtilis spores.

Germinated spores of Bacillus subtilis were incubated in outgrowth medium and tested periodically for capacity to sporulate when suspended in sporulation medium. Concurrent measurements were made of deoxyribonucleic acid (DNA) content and numbers of cell division septa and nucleoids. Sporulation potential is shown to reach a peak at about 110 min at which time the chromosomes are probably well into the second round of replication. Experiments with nalidixic acid show that sporulation potential can be generated in the outgrowth medium even when DNA synthesis is largely prevented. Further experiments show that nalidixic acid apparently does not prevent the formation of DNA initiation complexes, which can subsequently function after resuspension in the sporulation medium. The results support those previously obtained with a temperature-sensitive DNA mutant which indicated that sporulation could only be induced at a specific stage of chromosome replication, and then only if the cells are in a state of nutritional "step-down".