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.     

拟南芥黄心突变体yh基因的图位克隆及分析

在研究光合作用相关基因的过程中,获得了一个叶片为黄心(yellow heart,yh)的突变株,与野生型拟南芥(Col 0)相比,其新生叶片发黄,突变表型由隐性单基因控制。采用图位克隆及其精细定位技术,将yh突变基因定位在1号染色体的INS1_55_342与INS1_56_34区间,物理距离约为676 kb。通过测序得知yh在At1g64790第44个内含子剪接处有4个碱基的缺失,导致内含子剪切位点的变化。RT PCR分析显示,该基因表达降低,是At1g64790基因的一个新等位突变。研究表明,yh突变体与叶绿体的发育相关,可为进一步探究植物叶绿体和叶片发育机制提供新的遗传材料。     

Characterization of a spontaneous avirulent mutant of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> Serogroup 6: Evidence of DotA and flagellin involvement in the loss of virulence

The pathogenesis of Legionella pneumophila mainly resides in its ability to inhibit the phagosome-lysosome fusion, which normally prevents the killing of the host cells. In order to characterize the molecular alterations that occurred in a spontaneous avirulent mutant of Legionella pneumophila serogroup 6, named Vir-, we investigated the ability of the mutant to adhere to and multiply in the WI26VA4 alveolar epithelial cell line and in human macrophages, when compared to its parental strain, Vir+. We also determined the colocalization of bacteria with LAMP-1 to gain an insight into the phagosome-lysosome fusion process. Additionally, we determined the flagellin expression and dotA nucleotide sequencing. We observed a lack of expression of flagellin and an in-frame mutation in the dotA. gene. The data obtained strongly suggest the loss of virulence of the mutant could probably be due to the absence of flagellin and the dysfunctional type IV secretion System, resulting from the DotA protein being severely compromised.     

Amino Acid Substitutions and Intragenic Duplications of Bacillus sp. PS3 Flagellin Cause Complementation of the Bacillus subtilis Flagellin Deletion Mutant

Bacillus sp. PS3 produces a glycosylated flagellin. In this study, a number of the glycosylated residues of the flagellin protein were found to be located in the central variable region of this protein. We also report that the motility defect of the Bacillus subtilis flagellin mutant was complemented by Bacillus sp. PS3 flagellin variants without glycosylation, which contained amino acid substitutions and intragenic duplications in the variable region of flagellin.     

L1.LtrB内含子编码蛋白反转录结构域关键催化位点分析及功能验证

【目的】筛选影响Ll.LtrB内含子编码蛋白(Intron encoded protein,IEP)反转录功能的关键催化位点,并获得无反转录活性的IEP突变体。【方法】首先,利用NCBI数据库,通过序列比对及同源建模方法筛选影响IEP反转录功能的关键氨基酸催化位点;然后,对筛选获得的关键催化位点进行定点突变,同时以Targetron载体为模板,构建无反转录功能的突变型Targetron打靶系统;最后,以大肠杆菌lacZ基因为例,体内验证IEP突变体的功能及其对Ⅱ型内含子"归巢"效率的影响。【结果】筛选到C164和G214两个位点是影响内含子编码蛋白反转录功能的关键氨基酸残基,并获得C164K和G214W两个突变体。体内功能分析表明,此两个位点突变完全失活了Ⅱ型内含子的"归巢"功能。【结论】筛选并获得了失活反转录功能的Ll.LtrB内含子编码蛋白突变体,为深入研究Ⅱ型内含子的结构和"归巢"机理奠定了基础。     

Both the Extracellular Leucine-Rich Repeat Domain and the Kinase Activity of FLS2 Are Required for Flagellin Binding and Signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

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.     

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook‐associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non‐motile mutant cells that are unable to assemble flagellar filaments and pentagon‐shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella‐deficient mutants (i.e., in the hook, rod, or MS‐ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.     

Maf‐dependent bacterial flagellin glycosylation occurs before chaperone binding and flagellar T3SS export

Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non‐glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins. Received: 27 November 1997 / Accepted: 19 March 1998     

Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of <Emphasis Type="Italic">Arabidopsis</Emphasis> FLS2 exhibiting characteristically different perception specificities

Bacterial flagellin is known to stimulate host immune responses in mammals and plants. In Arabidopsis thaliana, the receptor kinase FLS2 mediates flagellin perception through physical interaction with a highly conserved epitope in the N-terminus of flagellin, represented by the peptide flg22 derived from Pseudomonas syringae. The peptide flg22 is highly active as an elicitor in many plant species. In contrast, a shortened version of the same epitope derived from Escherichia coli, flg15^{E coli}, is highly active as an elicitor in tomato but not in A. thaliana or Nicotiana benthamiana. Here, we make use of these species-specific differences in flagellin perception abilities to identify LeFLS2 as the flagellin receptor in tomato. LeFLS2 is most closely related to AtFLS2, indicating that it may represent the flagellin receptor of tomato. Expression of the LeFLS2 gene in Arabidopsis did not result in accumulation of its corresponding gene product, as indicated by experiments with LeFLS2-GFP fusions. In contrast, expression of LeFLS2-GFP fusions in N. benthamiana, a species that, like tomato, belongs to the Solanaceae, was obviously functional. N. benthamiana plants transiently expressing a LeFLS2-GFP fusion acquired responsiveness to flg15^{E coli} to which they are normally unresponsive. Thus, LeFLS2 encodes a functional, specific flagellin receptor, the first to be identified in a plant family other than the Brassicaceae. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Flagellin (FliC) protein sequence diversity among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> does not correlate with H serotype diversity

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensis fliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensis hag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensis fliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active β-lactamase fusion proteins (BlaM⁺) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line Katoill. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.     

海假交替单胞菌fliC-02330基因缺失影响生物被膜形成及厚壳贻贝幼虫附着变态

【背景】假交替单胞菌属是一种广泛分布于海洋环境的革兰氏阴性细菌,存在于海底沉积物中,能分泌大量的胞外产物形成海洋微生物被膜,从而诱导海洋无脊椎动物的附着。【目的】探究海假交替单胞菌鞭毛蛋白fliC基因对生物被膜形成及厚壳贻贝诱导活性的影响。【方法】通过基因敲除构建海假交替单胞菌fliC-02330基因缺失突变菌,研究突变菌和野生菌菌落形态、生物被膜形成能力、胞外物质以及对厚壳贻贝幼虫附着变态的诱导能力等的差异性。【结果】与野生菌相比,突变菌菌落表型出现褶皱,运动能力下降,形成被膜膜厚增加,以及对幼虫附着变态诱导活性下降。共聚焦扫描发现,fliC-02330基因缺失突变菌胞外多糖含量下降,而蛋白含量上升。【结论】海假交替单胞菌鞭毛蛋白fliC-02330基因缺失促进生物被膜形成,但抑制厚壳贻贝幼虫附着变态。本研究为探究细菌鞭毛蛋白基因与厚壳贻贝幼虫的作用机制,以及后续进一步探索微生物参与海洋无脊椎动物附着变态提供一定的理论依据。     

Innate immune recognition of flagellin limits systemic persistence of Brucella

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin‐deficient mutant of B. melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand‐off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.     

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.