| Genus Bacillus was comprised of Gram-positive and rod-shaped bacteria with low G+C and spore-forming.Many Bacillus species were important resources, because they could product spores and a variety of interesting components. The study on classification system of the genus Bacilluswas very important for exploring potential strains and promoting their application value. Several significant alterations has taken place in classification system of the genus Bacillus, due to improvement of classification method,change of classification index and increasing discovery of novel species.Fatty acids were important components of cell membrane of bacteria, and were subjected to the control of genetic material on cell membrane. The components and contents of fatty acids of Bacillus species were highly conserved and stable. More than 300 fatty acids have been found in bacteria. Differences in chain length,position of double bond and binding of functional group made fatty acids very useful as taxonomic markers. The cluster analysis of fatty acids could be used for determination of single bacterial species, which had superiority over traditional physiological characteristics. At present, Sherlock Microbial Identification System (Sherlock MIS) developed by MIDI company, USA, was a routine tool for fatty acid determination; it had a lot of advantages such as easy, inexpensive, rapid, sensitive characteristic, having a high degree of automation. Main steps of fatty acid analysis comprised bacterial culture, harvesting, saponification, methylation and extraction. The results of fatty acid determination were subjected to detection method, harvesting, culture temperature, time and medium component, etc. However, the Sherlock MIS built-in database had limited number of Bacillus species (only 25 species, accounting for 13.8% of the genus Bacillus) and updated very slowly, which was far from meeting the demand of Bacillus identification and classification.In order to construct a special phylogeny system of the genus Bacillus based on fatty acid, type strains of the genus Bacillus were obtained from DSMZ through international cooperation. The optimum detection scheme for the genus Bacillus determination was investigated by studying effects of different detection conditions on Sherlock MIS fatty acid results. Fatty acids of 90 type strains were assayed and analyzed by cluster analysis to construct a new type phylogeny system appropriate for the genus Bacillus. Meanwhile, searching novel species and screening functional strain of Bacillus were carried out based on this new phylogeny system. The main results of this study were summarized as follows:1. Study on the optimum detected conditions for fatty acids of the genus BacillusUsing type species as objects, effects of different detection conditions on fatty acid results of the genus Bacillus were investigated. Sherlock MIS were found to have shorter detection time (7 min) than GC/MS (45 min) under identical conditions. The optimum conditions for the detection were saponification time of 25 to 30 min, saponification reagent dosage of 1.0 mL, methylation reagent dosage of 1.5 mL, bacterial harvesting of 20 to 40 mg, bacterial culture temperature of 28?,bacterial culture time of 24 h and bacterial culture medium of Trypticase Soy Broth Agar (TSBA). Ten Bacillus type species were selected and repetition trials of their fatty acid detection were carried out by Sherlock MIS. A good reproducibility was observed in the trial and the tested bacteria were all accurately identified at Bacillus species levels.2. Phylogeny of the genus Bacillus based on fatty acid biomarkersAccording to the detected method described above, fatty acids of 90 type species of the genus Bacillus were extracted and 29 fatty acid biomarkers were obtained. The carbon lengths of those biomarkers ranged from 10 to 20. From statistical analysis, sum of relative percentage content of 6 largest of fatty acid biomarker were 15:0 anteiso,15:0 iso,17:0 anteiso,16:0,17:0 iso and 16:0 iso. Among which,15:0 anteiso and 15:0 iso possessed high content and were fully distributed type.17:0 anteiso,16:0,17:0 iso and 16:0 iso were middle content and not fully distributed type, and the rest belonged to low content and not fully distributed type. The 29 fatty acid biomarkers were divided into three classes by diversity analysis, namely high diversity high content, high diversity middle content and low diversity low content. Based on the distribution and diversity characteristics of fatty acid,10 parameters e.g.16:0 iso,16:0,17:0 iso,17:0 anteiso,15:0 iso,15:0 anteiso,15:0 iso/15:0 anteiso,17:0 iso/17:0 anteiso, diversity index [H] and evenness index(J) were selected to perform fatty acid phylogeny of the genus Bacillus.3. Biological characteristics of fatty acid groups of the genus BacillusNinety Bacillus species were divided into five fatty acid groups, such as Bacillus cereus group, Bacillus subtilis group, alkaliphilic group, acidophilus group and mesophilic group. After that, comparison between cluster analyses of phenetic classification and fatty acid classification were also conducted and it was found that both classification systems had identical result. Each fatty acid cluster group had special phonetic characteristics:the first group was aerobic, positive for gelatin reaction; the seconed group was positive for V-P reaction; the third group was facultative aerobic, alkaliphilic and positive for salicin reaction; the fourth was acidophilus and the fifth was facultative anaerobic, mesophilic and utilizing xylitol for energy. Therefore, fatty acid phylogenetic classification system of the genus Bacillus had a significance of physiological and biochemical characteristics, which the other classification did not have. This new type phylogenetic system was expected to become a new classification system.Representative species belonged to different fatty acid groups were selected to study biological characteristics. The results showed that Bacillus species of the first and fourth fatty acid group with moist colonies produced more content of 15:0 anteiso, while the second fatty acid group with dry colonies produced more unsaturated and 15:0 iso fatty acid. Molecular phylogeny based on 16S rRNA, gyrB and rpoB gene indicated that none of gene was suitable for phylogenetic analysis of all Bacillus species. Combining with fatty acid phylogeny, suitable gene could be fast selected for each fatty acid group of the genus Bacillus. gyrB and rpoBgene were suitable for the first group, while 16S rRNA was suitable for the third and fourth group. Besides, fatty acid phylogeny might also reflect the evolutionary relationship of Bacillus species. Bacillus species in the first fatty acid group was in moist habitat, the second was in alkaline, dry or high-temperature habitat and the fourth was in acid habitat.4. Identification and discovery of novel Bacillus species based on fatty acid phylogenyThree novel species of the genus Bacillus were preliminary discovered based on fatty acid phylogeny. After taxonomy experiments, the results demonstrated that they were indeed novel species of the genus Bacillus. All results were summarized as follows:A slightly-halophilic endospore-forming bacterium was isolated from the No.1 pit soil of Emperor Qin's Terracotta Warriors in Xi'an City, Shanxi Province, China. The novel isolate FJAT-13831T was rod-shaped, motile, Gram-stain-positive, catalase-and oxidase-positive, grew aerobically at 0-5%NaCl (w/v) (optimum 0%),15-45? (optimum 30?), pH 4.0-10.0 (optimum pH 7.0). The colonies were greyish-white, rough, flat, circular in the nutrient agar (NA). Phylogenetic analyses based on both 16S rRNA and gyrB (DNA gyrase B subunit gene) gene showed that the novel isolate FJAT-13831T falled into the genus Bacillus cluster, validated by significant bootstrap values. The similarity of 16S rRNA between novel isolate FJAT-13831T and the most closely reference species B. pseudomycoides DSM 12442T in the cluster was 99.72%, gyrB gene was 93.8% and ANI (the average nucleotide identity) of 2881 core genes was 91.47%. The DNA-DNA hybridization value between the novel isolate FJAT-138311 and phylogenetically related species of B. pseudomycoides DSM 12442T was 69.1%(less than 70%), which indicated that FJAT-13831T represented a distinct species. The DNA G+C content of FJAT-13831T was 36.5 mol%. The cell wall peptidoglycan contained meso-diaminopimelic acid. The predominant menaquinone is MK-7 (89%), MK-5 (8%) and MK-4 (2%) were minor components. The major fatty acids were 15:0 iso,17:0 iso,16:0,13:0 iso,15:0 anteiso and 15:1 iso w5c with values of 21.03,11.49, 9.83,7.66,7.39 and 5.12%, respectively. Based on these results, the isolate FJAT-138311 was recognized as a novel Bacillus species. The name of B. bingmayongensis (MS) was proposed for this organism. A novel gram-stain-positive,catalase-positive, oxidase-negative, endospore-forming, motile, rod-shaped, aerobic bacterium, designated FJAT-13985T, was isolated from rhizosphere soil of Mesona chinensis collected from Fuzhou, Fujian,China. The isolate grew at pH 5.7-9.0 (optimum pH 7.0) and at 20-45? (optimum 30?), in 0-2%(w/v) NaCl (optimum 0-1%). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain FJAT-13985T should be assigned to the genus Bacillus. It was most closely related to the type strains of Bacillus drentensis (16S rRNA sequence similarity 97.85%), Bacillus vireti (97.69%) and Bacillus novalis (97.58%), and DNA-DNA relatedness were 36.63,12.11 and 32.08%, respectively. The major cellular fatty acids are 15:0 anteiso and 15:0 iso. Strain FJAT-13985T, contained MK-7 as the predominant respiratory quinone. meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The genomic phylogenetic analysis, DNA-DNA relatedness values, phenotypic characteristics and chemotaxonomic data supported the view that strain FJAT-13985T represented a novel species of the genus Bacillus, for which the name B. mesonae (MS) was proposed.Among a large collection of Taiwanese soil isolates collected from taoyuan town,Taiwan, a novel Gram-stain-positive, rod-shaped, endospore-forming bacterial strain FJAT-14515T, was subjected to a polyphasic study including 16S rRNA sequence analysis, G+C content, cellular fatty acid composition analysis and comparative phenotypic characterization. The colonies were pale yellow, flat and edge irregularity. The bacterium could grow at 20-35? and the optimum growth temperature was at 30?. The suitable pH for FJAT-14515T were 6-9 (optimum 7.0), and the growth occurred (w/v) NaCl were in the range of 0-5%(optimum 0-1%).16S rRNA sequence similarities of FJAT-145151T with its closely related strains B. muralis and B. simplex were 97.55 and 97.48%, and DNA-DNA relatedness were 61.53% and 58%, respectively. The content of DNA G+C was 35 mol%. The major fatty acids were 15:0 anteiso (40.63%),15:0 iso (20.70%),16:0 (9.40%) and 14:0 iso (98%). All data supported that FJAT-14515T was a novel species belonging to the genus Bacillus, named B. cihuensis (MS).5. Study on functional Bacillus species based on fatty acidUsing Ralstonia solanacearum (pathogen of bacterial wilt disease) and Fusarium oxysporum f. sp. cubense (pathogen of banana wilt pathogen) as targets, biocontrol agents were screened from more than 300 Bacillus strains isolated from soil samples collected in China. It was found that strain FJAT-B had the strongest inhibition on R. solanacearum by the inhibition zone test. According to the colony morphology, physiology and biochemistry tests, comparisons of 16S rRNA and ITS sequences as well as fatty acid analysis, strain FJAT-B was identified as B. amyloliquefaciens. The optimum growth pH of FJAT-B was at pH 7-8. Its growth attached to logarithmic phase after 36 h, and gradually reduced by increasing salt concentration. Four Bacillus strains, designated as FJAT-4537, FJAT-4539, FJAT-4543 and FJAT-4544, were screened with effective suppression on F oxysporum f. sp. cubense on PDA agar. The four strains were identified as Paenibacillus polymyxa by fatty acid and 16S rRNA sequence analysis. They had similar biological characteristics and their main fatty acids were 15:0 anteiso. Using DPS statistic software, it was found that the inhibitory effect of biocontrol strains had negative and positive correlation with fatty acids 14:0 and 17:0 anteiso. The inhibitory effects of the four strains decreased with concentration reduction, very sensitive to temperature and not sensitive to UV. They had strong inhibitory effects at pH 9. The comprehensive results showed that strain FJAT-4539 had the strongest suppression against F. oxysporum f. sp. cubense. |
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