水稻内生成团泛菌YS19共质体形成差异表达蛋白MalE及其兼职功能

成团泛菌YS19是从水稻“越富”品种中分离的一种优势内生细菌,与宿主水稻互作时具有多种促生作用,其形成的共质体(symplasmata)结构与菌体抗逆及与宿主互作有重要意义．研究发现了一种在YS19共质体形成阶段高表达的差异蛋白,对其用肽指纹图谱进行鉴定,发现其属于周质空间麦芽糖结合蛋白家族．克隆了该蛋白质的基因,重组表达并分离纯化了该蛋白质,发现它是一种兼职功能蛋白,其不仅参与麦芽糖的ABC运输系统,而且在强酸环境下不易发生变性沉淀,并可通过疏水面的显著暴露结合底物蛋白来发挥分子伴侣活性,这些兼职功能构成了菌体抗逆生存适应性的重要分子基础．     

Rice endophyte Pantoea agglomerans YS19 forms multicellular symplasmata via cell aggregation

Pantoea agglomerans is characterized by the formation of multicellular symplasmata. One unanswered question regarding this bacterium is how these structures are formed. In this study, the rice diazotrophic endophyte P. agglomerans YS19 was selected for exploration of this theme. YS19 was labeled with green fluorescent protein and the resulting recombinant YS19::gfp was observed to grow only slightly more slowly (a decrease of 5.5%) than the wild-type strain, and to show high GFP label stability (label loss rate 8.9218 x 10(-6) per generation, nearly reaching the generally accepted spontaneous mutation rate for most bacteria). YS19::gfp resembled the wild-type YS19 in symplasmata formation and growth profiles. Based on associated cultivation of both strains by mixing their individually cultivated single cells, symplasmata were formed and composed of both YS19::gfp and YS19, suggesting that YS19 formed symplasmata via aggregation, not proliferation, of the original single cells.     

In vitro symplasmata formation in the rice diazotrophic endophyte Pantoea agglomerans YS19

Pantoea (formerly Enterobacter) agglomerans YS19 is an endophytic diazotrophic bacterium isolated from rice (Oryza sativa cv. Yuefu) grown in temperate climatic regions in west Beijing (China). The bacterium forms aggregate structures called `symplasmata'. A symplasmatum is a multicellular aggregate structure in which several (at least two) to hundreds of individual cells tightly bind together. The studies on the symplasmata formation of YS19 showed that there were two growth stages for YS19, including the single cell stage existing before exponential growth phase and the symplasmata forming stage starting at the early stationary growth phase in liquid GY (glucose yeast extract) medium or at the end of the exponential growth phase in liquid LB (Luria-Bertani) medium. There was a correlation between symplasmata formation and bacterial growth phase. When the medium was acidified, the cell growth rate was affected by the low pH of the medium, but the time required for symplasmata formation was not influenced by it. YS19 also formed symplasmata on agar medium, where more symplasmata were formed than in liquid medium. The volume of individual constitutional cells of symplasmata was sharply decreased by more than a half in comparison with that of the single cells existing before symplasmata formation. On all the media tested, YS19 formed symplasmata in most of the cell growth phases. The genome DNA/DNA homology between P. agglomerans YS19 and type strain P. agglomerans JCM1236^T (ATCC27155^T) was determined as 90.1%, confirming its membership of P. agglomerans. In order to investigate the phylogenetic relationships of YS19 at the intraspecific, intrageneric and super-generic level, the 16S rDNA similarities between strain YS19 and 17 other strains of Pantoea and 4 representatives of the closely related genera were analyzed. All the strains of Pantoea were clustered into 5 groups, and YS19 was clustered in a unique branch. The 16S rDNA similarity between YS19 and type strain JCM1236^T was 93.9%, much lower than the generally accepted value (=97%) for members of the same species, indicating that the 16S rDNA of YS19 has a distinct molecular characteristic.     

Colonization of endophyte <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> YS19 on host rice,with formation of multicellular symplasmata

Pantoea agglomerans YS19 is a diazotrophic endophyte isolated from rice (Oryza sativa cv. Yuefu) grown in a temperate-climatic region in west Beijing (China). The colonization of YS19 on host rice was studied in this paper. It was revealed that YS19 colonizes in all the tissues of rice seedlings, including roots (dominantly at elongation regions, lateral root junctions, root hairs and root caps), stems and leaves. More YS19 colonizes in stem and leaves (1.40 × 10⁵ CFU mg⁻¹ fresh weight) than that in roots (3.60 × 10⁴ CFU mg⁻¹). Symplasmata, a kind of adaptive structure of the strain for its endophytic living, were repeatedly observed to form inside root or stem cortex parenchyma tissues, as well as on leaf surfaces and also rhizoplanes. A novel matrix protein (SPM43.1) with its expression paralleling to the formation of symplasmata was captured, whose meaning in structural construction of symplasmata was also discussed.     

成团泛菌YS19 symplasmata结构对菌体抵抗逆境的作用

成团泛菌（Pantoea agglomerans）YS19是从水稻“越富”品种中分离出的优势内生菌,其所形成的共质体（symplasmata）是一种与生物薄膜（biofilm）类似的多细胞聚集体结构,但细胞间联系比biofilm更加紧密。研究symplasmata结构对成团泛菌YS19抵抗逆境的贡献,有助于阐释内生菌与植物的相互作用的适应性。比较研究了symplasmata结构与散生菌体对于蔗糖渗透压冲击、重金属离子和干燥处理的抵抗能力差异,结果表明,与以散生状态存在的菌体相比,在面临逆境时形成symplasmata结构的菌体抗逆存活能力显著增强。     

内生菌与植物的相互作用：促生与生物薄膜的形成

植物内生菌由于其独特的生态学地位而广受关注,近年来有关植物内生菌与宿主相互作用的研究取得了很大进展.本文综述了植物内生菌通过分泌促生物质、拮抗病原菌等实现与宿主共生互作,同时植物为内生菌提供适宜的黏附表面,使其形成以生物薄膜(biofilm)为主要形式的多细胞聚集体结构以更好地适应周围的生存环境,从而更加高效地对植物产生促生作用.本文论述了内生菌在与植物的互作中形成的多细胞聚集结构在抵抗非生物胁迫方面的独特生理及生态学意义,结合水稻内生成团泛菌YS19形成多细胞聚集体symplasmata现象及其生物学效应,对未来有关植物内生菌的研究方向提出了一些看法.     

In Vitro Adsorption Revealing an Apparent Strong Interaction Between Endophyte <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> YS19 and Host Rice

Pantoea (formerly Enterobacter) agglomerans YS19 is a dominant diazotrophic endophyte isolated from rice (Oryza sativa cv. Yuefu) grown in a temperate-climate region in west Beijing, China. In vitro adsorption and invasion of YS19 on host plant root were studied in this research. Adsorption of YS19 on rice seedling roots closely resembled the Langmuir adsorption and showed a higher adsorption quantity than the control strains Paenibacillus polymyxa WY110 (a rhizospheric bacterium from the same rice cultivar) and Escherichia coli HB101 (a general model bacterium). Adsorption dynamics study revealed high rates and a long duration of the YS19-rice root adsorption process. Adsorption of YS19 was mainly observed on the root hair, though which it enters the plant. This in vitro adsorption study revealed an apparent strong interaction between YS19 and rice at the early endophyte-host recognition stage.     

NMR assignment of intrinsically disordered self-processing module of the FrpC protein of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

The self-processing module (SPM) is an internal segment of the FrpC protein (P415–F591) secreted by the pathogenic Gram-negative bacterium Neisseria meningitidis during meningococcal infection of human upper respiratory tract. SPM mediates ‘protein trans-splicing’, a unique natural mechanism for editing of proteins, which involves a calcium-dependent autocatalytic cleavage of the peptide bond between D414 and P415 and covalent linkage of the cleaved fragment through its carboxy-terminal group of D414 to \(\epsilon\)-amino group of lysine residue within a neighboring polypeptide chain. We present an NMR resonance assignment of the calcium-free SPM, which displays characteristic features of intrinsically disordered proteins. Non-uniformly sampled 5D HN(CA)CONH, 4D HCBCACON, and HCBCANCO spectra were recorded to resolve poorly dispersed resonance frequencies of the disordered protein and 91 % of SPM residues were unambiguously assigned. Analysis of the chemical shifts revealed that two regions of the intrinsically disordered SPM (A95–S101 and R120–I127) have a tendency to form a helical structure, whereas the residues P1–D7 and G36–A40 have the propensity to adopt a \(\beta\)-structure.     

Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200

This study investigated the relative abundance of extracellular and cell wall associated proteins (exoproteome), cytoplasmic proteins (proteome), and related phenotypic traits of Lactobacillus plantarum grown under planktonic and biofilm conditions. Lactobacillus plantarum DB200 was preliminarily selected due to its ability to form biofilms and to adhere to Caco2 cells. As shown by fluorescence microscope analysis, biofilm cells became longer and autoaggregated at higher levels than planktonic cells. The molar ratio between glucose consumed and lactate synthesised was markedly decreased under biofilm compared to planktonic conditions. DIGE analysis showed a differential exoproteome (115 protein spots) and proteome (44) between planktonic and biofilm L. plantarum DB200 cells. Proteins up‐ or downregulated by at least twofold (p < 0.05) were found to belong mainly to the following functional categories: cell wall and catabolic process, cell cycle and adhesion, transport, glycolysis and carbohydrate metabolism, exopolysaccharide metabolism, amino acid and protein metabolisms, fatty acid and lipid biosynthesis, purine and nucleotide metabolism, stress response, oxidation/reduction process, and energy metabolism. Many of the above proteins showed moonlighting behavior. In accordance with the high expression levels of stress proteins (e.g., DnaK, GroEL, ClpP, GroES, and catalase), biofilm cells demonstrated enhanced survival under conditions of environmental stress.     

Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism

Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopH^CBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH₂. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopH^CBD state has an elevated affinity for SycH₂ and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH₂ passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.     

Identification of DnaJ-like chaperone in Clostridium botulinum type A

Clostridium botulinum type A cells, when challenged to elevated temperature (45°C), increased the expression of at least nine heat shock proteins (HSPs). Simultaneously with the induction of HSPs, changes in the synthesis rates of other cellular proteins were observed. A 40-kDa stress protein was induced and its synthesis rate was enhanced when the cells were shifted to 45°C. Using heterologous antibodies raised against E. coli DnaJ heat shock proteins, the 40-kDa stress protein of C. botulinum type A has been identified as a DnaJ-like chaperone. The DnaJ chaperone might be involved in translocation of the neurotoxin and other cellular proteins across the cell membrane, repair of damaged proteins, and organism survival inside the host. This is the first report of the existence of a DnaJ-like chaperone in this organism.     

Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2

Pseudomonas aeruginosa is a pathogenic bacterium widely investigated for its high incidence in clinical environments and its ability to form strong biofilms. During biofilm development, sessile cells acquire physiological characteristics differentiating them from planktonic cells. But after treatment with disinfectants, or to ensure survival of the species in hostile environments, biofilm cells can detach. This complicates disinfection procedures. This study aimed to physiologically characterize cells detached from a P. aeruginosa biofilm and to compare them with their sessile and planktonic counterparts. We first tested planktonic growth kinetics and capacities to form new biofilms. Then we investigated cell-surface properties. And finally, we tested in vitro susceptibility to antibiotics. The results first indicated that sessile and detached cells have similar planktonic growth kinetics and cell-surface properties, distinguishable from those of planktonic cells. Interestingly, the three populations exhibited different biofilm-forming capacities, suggesting that there is a transitional phenotype between sessile and planktonic states, at least during the first hours following cell detachment. It is important to consider this observation when developing treatments to optimize disinfection processes. Surprisingly, the three populations showed the same antibiotic susceptibility profile.     

MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, <Emphasis Type="Italic">Methanothermobacter thermoautotrophicum</Emphasis> involving in stress response

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50°C) and high (70°C) growth temperatures than under the optimal growth temperature for the organism (65°C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4°C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0°C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.     

Illustration of HIV-1 protease folding through a molten-globule-like intermediate using an experimental model that implicates alpha-crystallin and calcium ions

The folding of HIV-1 protease to its active form involves the coordination of structure formation and dimerization, which follows a hierarchy consisting of folding nuclei spanning from the active site, hinge region, and dimerization domain. However, the biochemical characteristics of the folding intermediates of this protein remain to be elucidated. In an experimental model, the denaturation of the tethered dimer of HIV-1 protease by guanidine hydrochloride revealed an alternative conformation resembling the molten-globule state. The molten-globule state binds to the molecular chaperone alpha-crystallin and prevents its aggregation; however, the chaperone alone failed to reconstitute HIV-1 protease into its active form. Calcium ion assisted in the release of active enzyme from the chaperone complex. Alpha-crystallin, a member of the small heat-shock protein, assists proteins to fold correctly; however, the underlying principle of signals responsible for chaperone-mediated protein folding remains enigmatic. X-ray photoelectron spectroscopy has been employed to provide the evidence of calcium binding to alpha-crystallin and to decipher the effect of calcium binding on the chaperone-mediated refolding of HIV-1 protease. On the basis of our spectroscopic data, we propose that calcium ions interact with the carboxyl groups of the surface-exposed acidic amino acids of alpha-crystallin bringing electrostatic interference, which plays a pivotal role in inducing conformational changes in the chaperone responsible for the release of the active enzyme.     

Extracellular HSP90: Conquering the cell surface

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone, assisting intracellularly in the folding and conformational regulation of a multitude of client proteins that play a crucial role in growth, cell survival and developmental processes(1). Moreover HSP90 interacts with a great number of molecules that are involved in the development and/or survival of cancer cells, allowing mutant proteins to retain or gain function while permitting cancer cells to tolerate the imbalanced signaling that such oncoproteins create (2,3). Prime examples include the HER-2 receptor, c-Raf-1, Akt/PKB, CDK4, and mutant p53 (4,5). Highly specific inhibitors of HSP90 have been identified and are currently under clinical evaluation. These include geldanamycin and its derivatives 17-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxygeldanamycin, which inhibit cancer cell proliferation in vitro and tumor growth in vivo (6-9).     

Visualization and functional analysis of the oligomeric states of <Emphasis Type="Italic">Escherichia coli</Emphasis> heat shock protein 70 (Hsp70/DnaK)

The molecular chaperone DnaK binds to exposed hydrophobic segments in proteins, protecting them from aggregation. DnaK interacts with protein substrates via its substrate-binding domain, and the affinity of this interaction is allosterically regulated by its nucleotide-binding domain. In addition to regulating interdomain allostery, the nucleotide state has been found to influence homo-oligomerization of DnaK. However, the architecture of oligomeric DnaK and its potential functional relevance in the chaperone cycle remain undefined. Towards that goal, we examined the structures of DnaK by negative stain electron microscopy. We found that DnaK samples contain an ensemble of monomers, dimers, and other small, defined multimers. To better understand the function of these oligomers, we stabilized them by cross-linking and found that they retained ATPase activity and protected a model substrate from denaturation. However, these oligomers had a greatly reduced ability to refold substrate and did not respond to stimulation by DnaJ. Finally, we observed oligomeric DnaK in Escherichia coli cellular lysates by native gel electrophoresis and found that these structures became noticeably more prevalent in cells exposed to heat shock. Together, these studies suggest that DnaK oligomers are composed of ordered multimers that are functionally distinct from monomeric DnaK. Thus, oligomerization of DnaK might be an important step in chaperone cycling.     

Physical map and dynamics of the chaperone network in Escherichia coli

Diverse families of molecular chaperones cooperate to effect protein homeostasis, but the extent and dynamics of direct interactions among chaperone systems within cells remain little studied. Here we used fluorescence resonance energy transfer to systematically map the network of pairwise interactions among the major Escherichia coli chaperones. We demonstrate that in most cases functional cooperation between chaperones within and across families involves physical complex formation, which pre-exists even in the absence of folding substrates. The observed connectivity of the overall chaperone network confirms its partitioning into sub-networks that are responsible for de novo protein folding and maturation and for refolding/disaggregation of misfolded proteins, respectively, and are linked by the Hsp70 system. We further followed heat-induced changes in the cellular chaperone network, revealing two distinct pathways that process heat-denatured substrates. Our data suggest that protein folding within cells relies on highly ordered and direct channelling of substrates between chaperone systems and provide a comprehensive view of the underlying interactions and of their dynamics.