The environment is extremely variable, since temperature, pH and osmolarity change rapidly and after morphogenetic pathway or bacteria. In addition the supply or nutrients usually undergoes drastic fluctuations. Those conditions cause defects/alteration on sporulation and germination of these spores by creating changes in genetic make up. The work being discussed here address sporulation and germination of Bacillus spores, isolated from polluted and saline areas (soils, plants). Initially 253 spore forming bacterial strains were isolated, among them 61 Bacillus strains showed negative germination response were selected. All the germination defective spore forming strains belonged to family Bacillacea. To illicit the defect in germination/sporulation pathway these strains were analyzed for their germination potential in nutrient (ALA, AGFK) and non-nutrient systems, with different germinants ( Nutrient - alanine, asparagine, isoleucine, valine, glutamine, proline phenylalanine and non-nutrient - CaOPA, MgOPA, CaC12, MgCl2, DPA) and without germinants.
Spores of Bacillus sp. Gd49, Gdl26 did not germinate in ALA while Gdl8 and Gd97 were deficient to germinate in AGFK system with any of germinant. Spores of Gd04, Gd97, Gd188, Gd192, Gd207 in ALA with alanine and spores or Gd207, Gd231 do not germinate in AGFK with asparagine. Spores of Gd49, Gd126 (do not germinate in ALA) Gd04, Gd97, Gd188, Gd192, Gd207 (do not germinate in ALA with alanine) Gd18, Gd97 (do not germinate in AGFK) Gd207 and Gd231 (do not germinate in AGFK with asparagine) germinate in CaOPA (Gd49), MgOPA (Gd04, Gd49, Gd188, Gd192, Gd231), CaCl2 (Gd188, Gd192, Gd23 I ), MgC12(Gd97, Gd126, Gd192, Gd207, Gd231) and DPA (Gd97, Gd188, Gd192). Probably the strains which were unable to germinate in ALA and AGFK system with alanine and asparagine respectively may have mutation in gerA, gerB, gerK and/or gerF.
Spores of some strains gave no germination with alanine using as sole germinant in ALA while with different substituted germinanls they respond at some extent such as Gd04 (aspargine, isoleucine, valine, glutamine), Gd97 (asparagines, praline, phenylalanine). Gd 188 (phenylalanine), Gd192 (glutamine)Gd207 (asparagines, isoleucine, valine. glutamine, proline. phenylalanine). Which exhibit that role or gerA / gerB. gerK if become defected then might bypass by using alternative nutrient germinants may be with other nutrient receptors or may be with some nutrient receptors.
In addition to nutrient-induced germination, a germination response is the rapid germination of spores by I:I chelate of Ca+2 and DPA. There are six groups on basis of germination responses to different non-nutrient germinants. First group consists of strains Gd18, Gd87, Gd92, Gd161, Gd193. Gd197, Gd213 which do not require any of no nutrient germinant to procede germination process. In second group different strains showed requirement of DPA (Gd55), MgOPA (Gd04, Gdl, Gd19, Gd85, Gd90, Gd181, Gd 208), both CaOPA, MgOPA (Gd49, Gd96) and with CaDPA + MgDPA + OPA (Od22) which exhibits the deficiency of endogenous DPA in strains of this group. Third group respond to MgC12, consisting of strains Gd05, Gd64, Gd 126, Gd207. Forth group consist on response of bacteria to two combinations CaDPA + MgC12 (Gd5) and MgDPA + CaC12 + MgC12 (Gd2. Gd214. Gd23 I. Gd233). Fifth group manifested response of bacteria to two combinations MgCI2 + DPA (Gd42, Gd97) and CaC12 + DPA (Gd99, Gd216, Gd233). Sixth group exhibited germination response to combination or MgDPA MgC12 I CaCI2 I DPA (Gd 16. (GD129. GD173. GD192 GD201. GD220); MgDPA CaC12 + OPA (Gd51); MgOPA + MgCI2 + DPA (GdI60. GdI88); CaDPA + CaC12 + DPA (Gd171 + Gd174); CaDPA + MgDPA + MgCI2 + DPA (Gd06 + Gd52, Gd2(5); CaDPA + CaC12 + MgCI2 + DPA (Gd4. Gd32); CadPA + MgDPA + CaCI2 + DPA (GdI4, Gd31, Gd195, Gd212) and CaDPA + MgDPA + CaC12 + MgCI2 + DPA (Gd3, Gd27, Gd33, Gd37, Gd98, Gd162, Gd163, Gd190. Gd200).
Among those 6 I strains (which were checked for their germination behaviour) 18 strains (Gd27, Gd96, Gd92, Gd205, Gd29, Gd05, Gd87, Gd19, Gd5. Gd99. Gd160, Gd207, Gd171, Gd85, Gd22, Gd212, Gd4. Gd173) were selected on basis or their germination responses to different germinants for in depth studies. Gd27 showed maximum germination with alanine in AGFK system although there were also variable responses to all germinants in non-nutrient, ALA and AGFK system. Gd29 germinated maximum with isoleucine in AGFK system, germination responses were negative with CaDPA, valine, glutamine, proline and phenylalanine. Gd05 have maximum germination response with valine in AGFK system. germination was observed with all germinants in ALA (except asparagine) and AGFK system while in non-nutrient system it was germinated only with MgC12. Gd87 showed maximum germination response with glutamine in AGFK system there was germination with all germinants in ALA and AGFK system but no germination observed in non-nutrient system. Gd19 have maximum germination response with proline in AGFK system although there was also germination with all germinants in ALA and AGFK but no germination in non-nutrient system (except with MgDPA). Gd5 showed maximum germination response with phenylalanine in AGFK system, there was also germination with all germinants in ALA and AGFK system but in non-nutrient system they germinate only with CaDPA and MgC12. Gd160 showed maximum germination with alanine in ALA system, germination behaviour was observed with all germinants in all the three systems except with CaDPA and CaC12 in non-nutrient system. Gd171 maximally germinated with isoleuine in ALA system and all germinants in ALA, AGFK system and non-nutrient system (except MgDPA and MgCI2).(ldS5 showed maximum germination with valine in AI.A system. germination was observed with all germinants in ALA (except phenylalanine) and AGFK. system, while in non-nutrient system with MgDPA alone. Gd22 have maximum germination with glutaminc in ALA system. germination was with all germinants in A(.FK. ALA (except asparaginc. phenylalanine)and non-nutrient system (except CaC12. MgCI2). Gd212 have maximum germination with CaDPA in non-nutrient system, germination was observed with all three systems except MgCI2 in non-nutrient system. Gd173 have maximum germination response with DPA in non-nutrient system although germination was observed with all germinants in AGFK, ALA (except asparagine) and non-nutrient (except CaDPA) system. These strains showed inhibitory behaviour with HgCI2 more than with NaN3. Gd92 exhibited maximum germination response with asparagine in AGFK system. It have variable germination responses with all germinants in ALA and AGFK system while there was no germination in non-nutrient system. Gd207 have maximum germination response with asparagine in ALA system, it germinated with MgC12 and phenylalanine (AGFK) and all germinant in ALA system except with alanine. Both of these strains showed maximum inhibition with NaN3.
Gd96 showed maximum germination response to alanine in AGFK system but germinated with CaDPA and MgDPA in non-nutrient while with alanine, asparagine, glutamine, proline in ALA and with all germinants in AGFK system. Gd205 have maximum germination with asparagine in AGFK system and variable responses to all germinants in all the three system except with CaC12 in non-nutrient system. Gd99 have maximum germinution response with phenylalanine in AGIK system in ALA and AGFK system germination was observed with all germinants but in non-nutrient system germination was observed with CaCI2 and DPA only. Gd4 have maximum germination response with MgC12 in non nutrient system, germination was with all the three systems except with MgDPA in non-nutrient system. These strains have almost equal inhibition with NaN3 and HgC12.
All these strains are devoid of proteases (except Gd207) and have different wall composition ratio than normal spores of B. subtilis PY79. They have more quantity of diaminopimelic acid and less amounts of glucosamine and teichoic acid except of Gd22 which have glucosamine more than in normal spores of B. subtilis PY79. While studying polypeptide profile of germinating spores of these 18 strains geminating spores of Gd5, Gd27, Gd29, Gd96, Gdl60 and Gdl71 showed protein profiles both with and without germinant in germination systems which enhanced their germination responses. De novo synthesiz polypeptides were of molecular weight 57, 22.5, (in Gd5), 61.8, 57.8, 56.5, 55, 48,47 (in Gd27) 54, 51,48.9,45.8,45.5, (in Gd29) 68, 67, 61.5, 61, 58.9, 56.7, 54.5, 50, 48.5, (in Gd96) 47.8, 46, 33.8, 51.5 (in Gd160) and 27kDa (in Gd171) while some polypeptides were commonly synthesized such as 53.2 (Gd27, Gd96), 50.5 (in Gd27 and Gd29), 47.5 (in Gd29 and Gd96) and 53.5kDa (in Gd96 and Gd160) with relative germinant.With synthesis of new polypeptides, reduction of some polypeptides was recorded too such as 54, 53, 50,48 (in Gd5) 56, 54.5, 15, 12 (in Gd27). 57. 20 (in Gd29) 65.4, 61.7, 53.7,41,27,9 (in Gd96) 61, 21, 23 (Gd96) and 24 kDa (in GdI71). Commonly reduced polypeptides were 63 (in Gd5 and Gd171).46 (in Gd5 and Gd27).66 (in Gd27, Gd29 and GdI60), 31 (in Gd27, Gd29 and Gd160) and 65 kDa (in Gdl60 and GdI71).
Gd5, Gd29, Gd92 and Gd96 were selected for illustrating structural defects via electron microscopy. Some defective structures in spore cell layers of four strains were observed. The overall impact of this work manifested that biochemical changes in cell wall constituents, alteration in protein products and deficiencies in proteases coordinated with defects and abnormalities in spore structure ultimately reflected in impairment in germination. Further molecular level studies arc required to illustrate the morphogenetic impairment in these strains.
16S rRNA similarities of four strains Gd5, Gd29, Gd92, and Gd96 aligned them with B. fusiformis. B. subtilis. B. subtilis and B cereus, respectively.