Cloning and Characterization of Flagellin Genes and Identification of Flagellin Glycosylation from Thermophilic Bacillus Species

Flagellin glycosylation was identified in Bacillus sp. PS3 and Geobacillus stearothermophilus. In vivo complementation showed that these flagellin genes did not restore the motility of a Bacillus subtilis flagellin mutant, whereas the genes encoding non-glycosylated flagellin from Geobacillus kaustophilus and Bacillus sp. Kps3 restored motility. Moreover, four types of flagellins expressed in B. subtilis were not glycosylated. We speculate that glycosylation is required for flagellar filament assembly of these bacilli.     

Temperature-Dependent Self-Splicing Group I Introns in the Flagellin Genes of the Thermophilic Bacillus Species

A previous report described the presence of a self-splicing group I intron in a flagellin gene from a thermophilic Bacillus species. Here, we present evidence that the splicing reaction of the flagellin introns is dependent on temperature. Furthermore, a complementation analysis using a Bacillus subtilis flagellin-deficient mutant indicated that the intron-containing flagellin gene significantly restored the motility of the mutant at higher temperatures.     

The Synthesis In Vitro of Flagellin by a Cell-Free Extract from Bacillus Pumilus

Abstract

Bacterial flagella are constructed mainly, or perhaps exclusively, of protein subunits, the flagellins. Demonstration is given in this report for the in vitro incorporation of radioactive amino acids into flagellin, it also appears that part of such incorporation reflects de novo synthesis of flagellin moleculus. Cell-free extracts were prepared from flagellated cells of Bacillus pumilus, by digestion of the cell wall with lysozyme, lysis in the Standard buffer of NIRENBERG and MATTHAEI (1961), treatment with deoxyribonuclease and centrifugation at 15.000×g. The reaction mixtures contained the cell-free extract, one or more [¹⁴C]-amino acids and the usual components required for cell-free protein synthesis. After incubation at 37° carrier flagellin was added and the pH of the reaction mixture adjusted to 2. Flagellin, which is soluble at this pH, was purified by disc electrophoresis or by reconstitution of flagellar filament at pH 5.4 followed by electrophoresis on a column of ethanolized cellulose. When an amino acid absent from B. pumilus flagellin (such as tyrosine) was used, the amount of radioactivity incorporated into the flagellin fraction was negligible as compared to that incorporated when radioactive leucine, arginine and lysine were used. The identity of the purified radioactive protein was established more conclusively by tryptic digestion and chromatographic separation of the resulting peptides. The ninhydrin positive peaks were shown to be coincident with the radioactive peaks. The radioactive peaks disappeared when a cell-free extract from non-flagellated mutant cells was used. The incorporation of radioactive methionine in the N-terminal position of the molecule indicated that at least some of the molecules had been synthesized de novo.     

Biochemical and Immunological Analyses of the Flagellin of Clostridium tyrobutyricum ATCC 25755

The monoclonal antibody 21E7-B12 (IgG₃) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and é-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.     

Identification of the Flagellin Glycosylation System in Burkholderia cenocepacia and the Contribution of Glycosylated Flagellin to Evasion of Human Innate Immune Responses

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-d-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.     

Sequence Diversity of the Bacillus thuringiensis and B. cereus Sensu Lato Flagellin (H Antigen) Protein: Comparison with H Serotype Diversity

We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagellin allele nucleotide sequences for 10 additional strains were retrieved from GenBank for a total of 106 Bacillus species and strains used in this study. These included 82 B. thuringiensis strains from 67 H serotypes, 5 B. cereus strains, 3 Bacillus anthracis strains, 3 Bacillus mycoides strains, 11 Bacillus weihenstephanensis strains, 1 Bacillus halodurans strain, and 1 Bacillus subtilis strain. The first 111 and the last 66 amino acids were conserved. They were referred to as the C1 and C2 regions, respectively. The central region, however, was highly variable and is referred to as the V region. Two bootstrapped neighbor-joining trees were generated: a first one from the alignment of the translated amino acid sequences of the amplified internal sequences of the hag alleles and a second one from the alignment of the V region amino acid sequences, respectively. Of the eight clusters revealed in the tree inferred from the entire C1-V-C2 region amino acid sequences, seven were present in corresponding clusters in the tree inferred from the V region amino acid sequences. With regard to B. thuringiensis, in most cases, different serovars had different flagellin amino acid sequences, as might have been expected. Surprisingly, however, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. Likewise, serovars from the same H serotypes were most often found clustered together, with exceptions. Indeed, some serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. Species-wise, B. halodurans, B. subtilis, and B. anthracis formed specific branches, whereas the other four species, all in the B. cereus sensu lato group, B. mycoides, B. weihenstephanensis, B. cereus, and B. thuringiensis, did not form four specific clusters as might have been expected. Rather, strains from any of these four species were placed side by side with strains from the other species. In the B. cereus sensu lato group, B. anthracis excepted, the distribution of strains was not species specific.     

Flagellin from Listeria monocytogenes is glycosylated with beta-O-linked N-acetylglucosamine

Glycan staining of purified flagellin from Listeria monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b suggested that the flagellin protein from this organism is glycosylated. Mass spectrometry analysis demonstrated that the flagellin protein of L. monocytogenes is posttranslationally modified with O-linked N-acetylglucosamine (GlcNAc) at up to six sites/monomer. The sites of glycosylation are all located in the central, surface-exposed region of the protein monomer. Immunoblotting with a monoclonal antibody specific for beta-O-linked GlcNAc confirmed that the linkage was in the beta configuration, this residue being a posttranslational modification commonly observed in eukaryote nuclear and cytoplasmic proteins.     

Expression of small RNAs by Bacillus sp. strain PS3 and B. subtilis cells during sporulation

A small RNA sequence identified in an rRNA-tRNA cluster from the thermophilic Bacillus sp. strain PS3 was examined. An oligonucleotide probe specific for the RNA bound to multiple restriction fragments in Bacillus sp. strain PS3 DNA, thus several copies of this sequence occur in its genome. Similar findings were observed using DNA from B. subtilis, B. stearothermophilus, Escherichia coli, Staphylococcus aureus, Haemophilus influenzae and Thermus thermophilus. This sequence apparently is widespread in the eubacteria. Northern analysis of RNA from sporulating Bacillus sp. strain PS3 and B. subtilis cells revealed RNA species homologous to the probe in both bacteria. Expression of the small RNA in B. subtilis depended on σ^H.     

Cloning of a gene for maltohexaose producing amylase of an alkalophilic Bacillus and hyper-production of the enzyme in Bacillus subtilis cells

Summary The gene for maltohexaose producing amylase from an alkalophilic bacterium, Bacillus sp. # 707, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a Bacillus subtilis plasmid pUB110, designated the resulting plasmids as pTUE306 and pTUB812, respectively. A common DNA region of approximately 2.5 kb was defined among the inserted DNAs. The enzymatic activity was lost when a part of the common region was deleted. The plasmids were stably maintained and the gene was well expressed in the bacterium, B. subtilis[pTUB812] which produced more than 70 times higher activity in the culture medium than did Bacillus sp. # 707. The major product of hydrolysis of starch by the enzymes of B. subtilis[pTUB812] and E. coli[pTUE306] was maltohexaose. The cloned gene corresponded to one of the genes for five components of malto-oligosaccharide-producing amylases of Bacillus sp. # 707.Abbreviations G1, G2, G3, G4, G5, and G6 glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose, respectively - kb kilobase pairs - kdal kilodalton - [] designated plasmid-carrier state     

Cloning of the cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. A2-5a and analysis of the raw starch-binding domain

The cyclodextrin glucanotransferase (CGTase) gene of alkalophilic Bacillus sp. A2-5a was cloned and expressed in Bacillus subtilis ANA-1 as a host. The DNA region included an open reading frame encoding a 704-amino-acid polypeptide with a typical raw starch-binding motif in its C-terminal region. The CGTase purified from Bacillus sp. A2-5a bound to raw starch as strongly as porcine pancreas α-amylase, as expected from the sequence motif. A chromosomal region (a DNA fragment of about 14.1 kbp) including the CGTase gene was also cloned and the nucleotide sequence was determined. Possible cyclodextrinase and putative cyclodextrin-binding protein genes were found in the flanking region of the CGTase gene, which implied that the novel starch-degradation pathway postulated for a gram-negative bacterium [Klebsiella oxytoca; Fiedler et al. (1996) J Mol Biol 256: 279–291] also exists in a gram-positive bacterium i.e. Bacillus. Received: 6 August 1999 / Received last revision: 8 October 1999 / Accepted: 22 October 1999     

Characterization of chitinase‐producing Serratia and Bacillus strains isolated from insects

Chitinases (EC 3.2.1.14) are enzymes that hydrolyze chitin by cleaving β‐1,4 N‐glycosidic bonds. These enzymes have been used for multiple applications in biotechnology, especially for controlling insect pests and phytopathogenic fungi. In the present study, we isolated two chitinase‐producing bacteria strains from insects (strain SCH‐1 from Moechotypa diphysis and strain SCH‐2 from Sphedanolestes impressicollis). Serratia sp. SCH‐1 was a short, rod‐shaped facultative anaerobe, while Bacillus strain SCH‐2 was a rod‐shaped endospore‐forming anaerobe. Strains SCH‐1 and SCH‐2 were identified as Serratia sp. and Bacillus sp., respectively based on 16S rRNA gene sequencing. Strain SCH‐1 shared maximum homology (99.44%) with Serratia nematodiphila DZ0503SBS1 and Serratia marcescens subsp. sakuensis KRED. Strain SCH‐2 had a maximum homology of 99.24% with Bacillus thuringiensis ATCC 10792 and Bacillus toyonensis BCT‐7112. Serratia sp. SCH‐1 contained greater levels of saturated fatty acids, but the concentration of branched acids, especially iso‐C_15:0, was highest in Bacillus sp. SCH‐2. Serratia sp. SCH‐1 possessed chitinase activity of 1.59 unit/mg protein after 5 days of incubation in culture medium. In contrast, Bacillus sp. SCH‐2 had a maximum activity of 0.84 unit/mg protein after 4 days of incubation. Chitinase isozymes produced by Serratia sp. SCH‐1 appeared as five bands with sizes of 20, 26, 36, 45 and 54 kDa. Bacillus sp. SCH‐2 showed a chitinase isozyme profile with three bands having sizes of 36, 45 and 50 kDa on SDS‐PAGE gels.     

Sequence Diversity of Bacillus thuringiensis Flagellin (H Antigen) Protein at the Intra-H Serotype Level

In Bacillus thuringiensis, the hag gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Specific flagellin amino acid sequences have been correlated to specific B. thuringiensis H serotypes, H1 to H67. Ten H serotypes, however, contain three or more antigenic subfactors, labeled a, b, c, d, or e, and have been subdivided into 23 serovars. In the present study, we set out to analyze the sequence diversity of flagellins among serovars from the same H serotypes. We studied the hag genes in 39 B. thuringiensis strains representing the 23 serovars from the 10 H serotypes mentioned above. A serovar and a biovar from an 11th H serotype were also included. The hag genes were amplified and cloned and their nucleotide sequences were determined and translated into amino acid sequences, or the sequences were retrieved directly from GenBank when available. Strains of the H3 serotype contained two or three copies of the fla gene, an ortholog of the hag gene. Strains of the H6 serotype contained three copies. Strains of all other H serotypes each contained a single copy of the hag gene. Alignments of amino acid sequences from all copies in all strains of the H3 serotype revealed short signature sequences, GGAG and SGG, GPDPDDAVKNLT, and DITTTK, that appeared to be specific to the H3c, H3d, and H3e antigenic subfactors, respectively. Similar short signature sequences, GDIT, AFIK, TSAGKA, and SAPSKG, were revealed for H8b, H8c, H20b, and H20c, respectively. Amino acid sequences in the flagellin central variable region were highly conserved among serovars of the H3, H5, H11, and H20 serotypes and much more divergent among serovars of the H4, H10, H18, H24, and H28 serotypes. Two bootstrapped neighbor-joining trees were respectively generated from the alignments of the amino acid sequences translated from all copies of the hag genes in the B. thuringiensis strains of the H3 and H6 serotypes. Sequence identities and relationships were revealed. A third bootstrapped neighbor-joining tree was generated, this one from the alignment of the flagellin amino acid sequences from all the B. thuringiensis strains in the study. Eight clusters, I to VIII, were revealed. Although most clusters contained strains and serovars from the same H serotype, clusters VII and VIII contained serovars from different H serotypes.     

Establishment of Monoclonal Antibodies Specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Endophytic Colonization of Balloon Flower by Antifungal Strain Bacillus sp. CY22

Endophytic Bacillus sp. CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci. Biotechnol. Biochem., 66, 1270-1275 (2002)). Three-month-old balloon flower seedlings were inoculated with 10⁷ cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later. After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy. More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp. CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root. Also, Bacillus sp. CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A. In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp. CY22R3 was inoculated into the soil.     

Isolation and characterization of four novel Gram-positive bacteria associated with the rhizosphere of two endemorelict plants capable of degrading a broad range of aromatic substrates

Four new Gram-positive, phenol-degrading strains were isolated from the rhizospheres of endemorelict plants Ramonda serbica and Ramonda nathaliae known to exude high amounts of phenolics in the soil. Isolates were designated Bacillus sp. PS1, Bacillus sp. PS11, Streptomyces sp. PS12, and Streptomyces sp. PN1 based on 16S rDNA sequence and biochemical analysis. In addition to their ability to tolerate and utilize high amounts of phenol of either up to 800 or up to 1,400 mg l⁻¹ without apparent inhibition in growth, all four strains were also able to degrade a broad range of aromatic substrates including benzene, toluene, ethylbenzene, xylenes, styrene, halogenated benzenes, and naphthalene. Isolates were able to grow in pure culture and in defined mixed culture on phenol and on the mixture of BTEX (benzene, toluene, ethylbenzene, and xylenes) compounds as a sole source of carbon and energy. Pure culture of Bacillus sp. PS11 yielded 1.5-fold higher biomass amounts in comparison to mixed culture, under all conditions. Strains successfully degraded phenol in the soil model system (2 g kg⁻¹) within 6 days. Activities of phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase were detected and analyzed from the crude cell extract of the isolates. While all four strains use ortho degradation pathway, enzyme indicative of meta degradation pathway (catechol 2,3-dioxygenase) was also detected in Bacillus sp. PS11 and Streptomyces sp. PN1. Phenol degradation activities were induced 2 h after supplementation by phenol, but not by catechol. Catechol slightly inhibited activity of catechol 2,3-dioxygenase in strains PS11 and PN1.     

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic Bacterium Geobacillus stearothermophilus

A group I intron that can be spliced in vivo and in vitro was identified in the flagellin gene of the thermophilic bacterium Geobacillus stearothermophilus. We also found one or two intervening sequences (IVS) of flagellin genes in five additional bacterial species. Furthermore, we report the presence of these sequences in two sites of a highly conserved region in the flagellin gene.     

Posttranslational Modification of Flagellin FlaB in Shewanella oneidensis

Shewanella oneidensis is a highly motile organism by virtue of a polar, glycosylated flagellum composed of flagellins FlaA and FlaB. In this study, the functional flagellin FlaB was isolated and analyzed with nano-liquid chromatography-mass spectrometry (MS) and tandem MS. In combination with the mutational analysis, we propose that the FlaB flagellin protein from S. oneidensis is modified at five serine residues with a series of novel O-linked posttranslational modifications (PTMs) that differ from each other by 14 Da. These PTMs are composed in part of a 274-Da sugar residue that bears a resemblance to the nonulosonic acids. The remainder appears to be composed of a second residue whose mass varies by 14 Da depending on the PTM. Further investigation revealed that synthesis of the glycans initiates with PseB and PseC, the first two enzymes of the Pse pathway. In addition, a number of lysine residues are found to be methylated by SO4160, an analogue of the lysine methyltransferase of Salmonella enterica serovar Typhimurium.     

Diazotrophic bacilli isolated from the sunflower rhizosphere and the potential of Bacillus mycoides B38V as biofertiliser

The nitrogen fixation by strains belonging to the Bacillus genus remains poorly explored. In this work, the diversity of endospore‐forming bacilli isolated from the rhizosphere of sunflower was evaluated. A total of 101 strains were identified based on the V1‐V2 variable regions of the 16S rRNA gene. Strains belonging to the genera Bacillus and Paenibacillus represented 41.6% and 58.4%, respectively, of total isolates. The production of indolic compounds was a common trait among the isolates, and approximately 75% of them exhibited positive nitrogenase activity; but only 9.2% displayed activities higher than 1 nmol C₂H₄ mg protein h^?1. Within the genus Bacillus, the isolates related to the B. cereus group displayed the highest nitrogenase activity and were the second most frequent group of Bacillus sp. isolated. Plants inoculated with the isolate B38V showed the highest N content, and their shoot dry weights were significantly increased compared with positive control. Our results indicated that B38V, which belongs to the B. mycoides species, has the potential to promote sunflower growth. The data obtained in this study provide additional information concerning the diversity of Gram‐positive diazotrophic within the genera Bacillus and Paenibacillus and their potential for the biofertilisation of sunflower crops.     

FliW and FliS Function Independently To Control Cytoplasmic Flagellin Levels in Bacillus subtilis

The cytoplasmic level of flagellin (called Hag) is homeostatically regulated in the Gram-positive bacterium Bacillus subtilis by a partner-switching mechanism between the protein FliW and either the Hag structural protein or CsrA, an RNA binding protein that represses hag translation. Here we show that FliW and the putative secretion chaperone FliS bind to Hag simultaneously but control Hag translation by different mechanisms. While FliW directly inhibits CsrA activity, FliS antagonizes CsrA indirectly by binding to Hag, enhancing Hag secretion, and depleting Hag in the cytoplasm to trigger the FliW partner switch. Consistent with a role for FliS in potentiating Hag secretion, the mutation of fliS crippled both motility and flagellar filament assembly, and both phenotypes could be partially rescued by artificially increasing the concentration of the Hag substrate through the absence of CsrA. Furthermore, the absence of FliS resulted in an approximately 30-fold reduction in extracellular Hag accumulation in cells mutated for CsrA (to relieve homeostatic control) and the filament cap protein FliD (to secrete flagellin into the supernatant). Thus, we mechanistically discriminate between the FliW regulator and the FliS chaperone to show that secretion disrupts flagellin homeostasis and promotes high-level flagellin synthesis during the period of filament assembly in B. subtilis.     

Endophytic Bacillus sp. Isolated from the Interior of Balloon Flower Root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C_15:0 (37%), C_17:0 (5.1%), and iso-C_15:0 (27.7%). DNA G + C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.