Isolation and Characterization of Outermost Layer Deficient Mutant Spores of Bacillus megaterium

Abstract

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by 'salt aggregation test,' which would facilitate selection of such mutants.The bacterial spores have chemically and morphologically specific structural components: the cortex, the coat, and the exosporium. Bacillus megaterium ATCC 12872 (QM B1551) spore has a thick outermost layer (OL) outside of the inner coat (IC) (2). This layer had been called "outer coat" by many including our group, but Gerhardt's group proposed to call it "exosporium" from its morphological and chemical features ( 11 ) . But it is not a typical exosporium as observed in B. cereus spores. We use the term "outermost layer" tentatively because terminology is not an essential problem in this paper. This layer is formed at the later stages of sporulation and is resistant to several treatments: sonic disintegration, lysozyme digestion, and alkali extraction (8). Our previous papers (15,16) showed that OL consists of a 'skelton' of phosphorylated galactosamine polymer carrying mannose in some parts and also contains peptides/proteins. But its function, biosynthesis, and especially morphogenesis, have not been clearly known. OL deficient mutant spore is a useful tool for elucidating these subjects. Koshikawa et al have isolated one OL deficient mutant (ATCC 33729) of this strain by chance selection (11), but only one mutant is not enough for this purpose so that we tried to isolate these mutant spores by taking advantage of the difference in density between the mutant and the parent spores.The parent strain, B. megaterium ATCC 12872 (QM B1551), was obtained from the American Type Culture Collection and the mutant, ATCC 33729, was supplied by Dr. Koshikawa at Setsunan University. According to the method of Ito and Spizizen (7) , the parent spores were treated with 0.45 M ethyl methanesulfonate 973

Bacillus anthracis Exosporium Protein BclA Affects Spore Germination, Interaction with Extracellular Matrix Proteins, and Hydrophobicity

“…The relative hydrophobicities of spores can be measured by a number of established techniques that include adherence to hydrocarbons (13,18,19,27), hydrophobic interaction chromatography (HIC) (7,10), salt aggregation (24), and contact angle measurements (14). To investigate the role of BclA in surface hydrophobicity, we compared spores with and without BclA (wild-type versus bclA mutant spores) in the bacterial adherence to hydrocarbon (BATH) assay as described previously by Rosenberg et al (18).…”

Characterization and Deposition of the Proteins in the Outermost Layer of Bacillus megaterium Spore

“…We reported that galactosamine-6-phosphate polymer begins to be deposited on the forespore from t9 (17) and may be responsible for the surface hydrophobicity of the spore (22). In this study, it was indicated that this sugar polymer does not participate in the deposition of P36 and P22, but may play the role in protecting these OL proteins against the releasing factor after deposition, because loss of these proteins was not observed in the mature spore and the forespore at tio of the wildtype strain where the sugar polymer had been deposited.…”

“…The suspension (20 mg dry weight/ml deionized water) of spore coat preparation (18) was sonicated at 30-sec intervals for a total sonication time of 3 min with the small probe of a Heat Systems Ultrasonics sonicator, model W-220F, at 4 C. After sonication, the suspension placed on the tube containing the ten stepwise Urografin density gradients (density range, 1.28-1.37 g/cm3) was centrifuged as previously described (22). Two bands of density, 1.28 and 1.35 g/cm3, respectively, were collected by a syringe, washed with deionized water several times after centrifugation (10,000 x g for 20 min), and lyophilized after dialysis against deionized water for three days.…”

“…B. megaterium ATCC 12872 was obtained from the American Type Culture Collection, and the OL deficient mutants, MAE02, MAE04, and MAE05, were isolated in our laboratory (22). Cells of these strains were grown and sporulated in SNB medium at 30 C as previously described (22). Phase-bright spores reach maximum at t8 (t, indicates n hr after the end of exponential growth) and this stage was used as an internal marker for the timing of developmental events.…”

“…All of the OL deficient mutant spores including MAE05 spore lack these OL proteins (22). Absence of these proteins in the mature spore does not necessarily mean no synthesis of them, because the mutation of the gene responsible for processing or assembly may be possible.…”