Kin-Fu Chak

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

BACKGROUND Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition. RESULTS The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 A resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel alpha/beta fold and contains a Zn2+ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (alpha1(*)-loop12-alpha2(*); where * represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23-alpha3(*)-loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7. CONCLUSIONS The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.

The crystal structure of the nuclease domain of colicin E7 suggests a mechanism for binding to double-stranded DNA by the H-N-H endonucleases.

The bacterial toxin ColE7 contains an H-N-H endonuclease domain (nuclease ColE7) that digests cellular DNA or RNA non-specifically in target cells, leading to cell death. In the host cell, protein Im7 forms a complex with ColE7 to inhibit its nuclease activity. Here, we present the crystal structure of the unbound nuclease ColE7 at a resolution of 2.1A. Structural comparison between the unbound and bound nuclease ColE7 in complex with Im7, suggests that Im7 is not an allosteric inhibitor that induces backbone conformational changes in nuclease ColE7, but rather one that inhibits by blocking the substrate-binding site. There were two nuclease ColE7 molecules in the P1 unit cell in crystals and they appeared as a dimer related to each other by a non-crystallographic dyad symmetry. Gel-filtration and cross-linking experiments confirmed that nuclease ColE7 indeed formed dimers in solution and that the dimeric conformation was more favored in the presence of double-stranded DNA. Structural comparison of nuclease ColE7 with the His-Cys box homing endonuclease I-PpoI further demonstrated that H-N-H motifs in dimeric nuclease ColE7 were oriented in a manner very similar to that of the betabetaalpha-fold of the active sites found in dimeric I-PpoI. A mechanism for the binding of double-stranded DNA by dimeric H-N-H nuclease ColE7 is suggested.

Involvement of Acidic Fibroblast Growth Factor in Spinal Cord Injury Repair Processes Revealed by a Proteomics Approach

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01–0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.

Localization and Characterization of a Gene on the ColE3-CA38 Plasmid that Confers Immunity to Colicin E8

Escherichia coli W3110 cells carrying the ColE3-CA38 plasmid are immune to externally added colicin E8, a newly described member of the E group colicins. By molecular cloning and transposon mutagenesis we localized the colicin E8 immunity gene between the EcoRI site (4.0 kb on the restriction map) and the PvuII site (3.68 kb) of the ColE3-CA38 plasmid. This placed the colicin E8 immunity gene between the colicin E3 immunity gene and lys, the region which determined mitomycin C sensitivity. Insertion of a transposon into the colicin E3 structural gene prevented the synthesis of active colicin and completely abolished mitomycin C sensitivity, but had no effect on the two immunity genes. In contrast, insertion of a transposon into the colicin E8 immunity gene had no effect upon colicin E3 production or colicin E3 immunity but did abolish mitomycin C sensitivity. The phenotype conferred by plasmids with a transposon inserted into the lys region of ColE3-CA38 was dependent upon the site of insertion.

The Critical Roles of Polyamines in Regulating ColE7 Production and Restricting ColE7 Uptake of the Colicin-producingEscherichia coli *

The ColE7 operon is an SOS response regulon, which encodes bacteriocin ColE7 to kill susceptible Escherichia coli and its related enterobacteria under conditions of stress. We have observed for the first time that polyamines confer limited resistance against ColE7 on E. coli cells. Thus, this study aims to investigate the role of polyamines in modulating the protective effect of the E. coli cells against colicin. In the experiments, we surprisingly found that endogenous polyamines are also essential for ColE7 production, and the rate of polyamine synthesis is directly related to the SOS response. Our experimental results further indicated that exogenous polyamines suppress the expression of TolA, BtuB, OmpF, and OmpC proteins that are responsible for ColE7 uptake. Moreover, two-dimensional gel electrophoresis revealed that the production of two periplasmic proteins, PotD and OppA, is increased in E. coli cells under ColE7 exposure. Based on these observations, we propose that endogenous polyamines may play a dual role in the ColE7 system. Polyamines may participate in initiating the expression of the SOS response of the ColE7 operon and simultaneously down-regulate proteins that are essential for colicin uptake, thus conferring a survival advantage on colicin-producing E. coli under stress conditions in the natural environment.

Dissection of cry gene profiles of Bacillus thuringiensis isolates in Taiwan.

On the basis of the newly revised nomenclature system of cry genes, the PCR amplification method has been adopted to resolve the cry gene combinations of 294 Bacillus thuringiensis isolates from five selected areas of Taiwan. Our results indicate that cry1 (especially cry1A + 1B + 1F) and cry2 were the most abundant cry genes in Taiwan. In contrast, cry3 and cry6 genes were detected only on Yang Ming Mountain, while the cry13 gene was found only on Snow Mountain. In addition, some distinctive combinations of cry genes were detected in distinct areas of Taiwan, such as cry1C,cry1D, cry1C + 1D, cry4, cry1 + 4, cry1 + 11, cry4 + 11, and cry1 + 4 + 11 in the Taipei area; cry1A + 1C + 1F in the Taichung area; cry1E and cry1A + 1B + 1I on Yang Ming Mountain; cry1 + 13, cry1 + 2 + 11, and cry1 + 2 + 13 on Snow Mountain; and cry1 + 5 and cry1 + 2 + 5 on Jade Mountain. These data clearly indicate that the distribution of cry gene combinations of B. thuringiensis isolates seems to be geographically related.

A novel role of ImmE7 in the autoregulatory expression of the ColE7 operon and identification of possible RNase active sites in the crystal structure of dimeric ImmE7.

Site‐specific cleavage of mRNA has been identified in vivo for the polycistronic colicin E7 operon (ColE7), which occurs between G and A nucleotides located at the Asp52 codon (GAT) of the immunity gene (ceiE7). In vitro, this specific cleavage occurs only in the presence of the ceiE7 gene product (ImmE7). The crystal structure of dimeric ImmE7 has been determined at 1.8 Å resolution by X‐ray crystallographic analysis. We found that several residues located at the interface of dimeric ImmE7 bear surprising resemblance to the active sites of some RNases. These results suggest that dimeric ImmE7 may possess a novel RNase activity that cleaves its own mRNA at a specific site and thus autoregulates translational expression of the downstream celE7 gene as well as degradation of the upstream ceaE7 mRNA.

Cloning of Two New cry Genes from Bacillus thuringiensis subsp. wuhanensis Strain

Abstract. With PCR products as probes, we have cloned two new cry-type genes from Bacillus thuringiensis subsp. wuhanensis. The deduced amino acid sequence of the first clone is 77.3% identical to Cry1Ga1. The deduced protein sequence of the second clone is 69.8–78.7% identical to that of Cry1B group. The nomenclature assignment of these two clones is, therefore, named Cry1Gb1 and Cry1Bd1, respectively. The Cry1Bd1 is toxic to Plutella xylostella larvae, and the Cry1Gb1 is toxic to Pieris rapae larvae.

Facile in vivo transfer of mutations between the Bacillus subtilis chromosome and a plasmid harbouring homologous DNA.

Transformation of Bacillus subtilis strains carrying the point mutations spoIIA42 or spoIIA69 by a plasmid, pHM2, carrying the spoIIA+ allele gave occasional recombinants in which the plasmid had acquired the chromosomal mutation. Transformation of a spo+ strain by these derivative plasmids gave occasional recombinants in which the chromosome of the recipient had acquired the plasmid-borne spoIIA mutation. Both types of behaviour were recE+-dependent. Mutations spoIIA42 and spoIIA69 fell into a single complementation group.

PAL31 may play an important role as inflammatory modulator in the repair process of the spinal cord injury rat.

Functional regeneration in a complete T8 transection model Cheng et al. (1996) and most recently, acidic fibroblast growth factor (aFGF; also known as FGF‐1) involved in the repair process of the spinal cord injury (SCI) rat Tsai et al. (2008 ) have been reported. To further reveal the mechanism of the repair process of SCI, in additionally, we have identified a 30 kDa specific protein kinase A substrate induced at 6 days after SCI. However, the induction of the transducing signal was reduced in samples treated with aFGF. The 30 kDa protein was purified and identified by mass spectrometry as a novel protein, PAL31. The results of immunohistochemical study showed that PAL31 is abundantly expressed in the epicenter of the injured spinal cord and colocalizes with ED1‐positive cells (macrophages) and CD8 T lymphocytes. Over‐expression of PAL31 in RAW 264.7 cells resulted in the down‐regulation of macrophage chemoattractant protein 1, inducible nitric oxide synthase, and signal transducer and activator of transcription‐1. However, knockdown of PAL31 by small interfering RNA seems to lead to apoptosis when the cells were treated with inflammatory inducers. These experimental results suggest that PAL31 may involve in the modulation of the inflammatory response and, at the same time, prevent apoptosis process of macrophage after SCI.