利福霉素生产菌产生钝化RifSV物质的分离及性质研究

利福霉素ＳＶ（简称ＲｉｆＳＶ）生产菌──地中海拟无枝菌酸菌在生物合成ＲｉｆＳＶ过程中,产生一种能钝化自身产物（ＲｉｆＳＶ）的物质．实验证实,该物质是由谷氨酸、天冬氨酸、赖氨酸及缬氨酸等１４种常见氨基酸组成的蛋白质．分子量（ＭＷ）约２．５×１０４Ｄ,等电点（ＰＩ）为５．７—６．１．在１００℃下加热１０ｍｉｎ,其活性丧失．作用于ＲｉｆＳＶ的最适ｐＨ值范围为７．４—８．６,最适温度为２９℃,初步证实该物质是一种酶（暂称利福霉素钝化酶）     

A New Equation to Estimate Glomerular Filtration Rate in Chinese Elderly Population

Background

We sought to develop a new equation to estimate glomerular filtration rate (GFR) in Chinese elderly population.

Methods

A total of 668 Chinese elderly participants, including the development cohort (n = 433), the validation cohort (n = 235) were enrolled. The new equation using the generalized additive model, and age, gender, serum creatinine as predictor variables was developed and the performances was compared with the CKD-EPI equation.

Results

In the validation data set, both bias and precision were improved with the new equation, as compared with the CKD-EPI equation (median difference, −1.5 ml/min/1.73 m² vs. 7.4 ml/min/1.73 m² for the new equation and the CKD-EPI equation, [P<0.001]; interquartile range [IQR] for the difference, 16.2 ml/min/1.73 m² vs. 19.0 ml/min/1.73 m² [P<0.001]), as were accuracies (15% accuracy, 40.4% vs. 30.6% [P = 0.02]; 30% accuracy, 71.1% vs. 47.2%, [P<0.001]; 50% accuracy, 90.2% vs. 75.7%, [P<0.001]), allowing improvement in GFR categorization (GFR category misclassification rate, 37.4% vs. 53.2% [P = <0.001]).

Conclusions

A new equation was developed in Chinese elderly population. In the validation data set, the new equation performed better than the original CKD-EPI equation. The new equation needs further external validations. Calibration of the GFR referent standard to a more accurate one should be an useful way to improve the performance of GFR estimating equations.     

Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening

Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.     

Molecular Cloning of Novel Cellulase Genes <Emphasis Type="Italic">cel</Emphasis>9A and <Emphasis Type="Italic">cel</Emphasis>12A from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> GXN151 and Synergism of Their Encoded Polypeptides

A thermophilic bacterial strain GXN151 which could degrade Avicel efficiently was isolated and identified as Bacillus licheniformis. A genomic library of GXN151 was constructed and two novel endoglucanase genes designated cel9A and cel12A were isolated by screening the library on carboxylmethyl cellulase indicator plates. The analysis of amino acid sequences deduced from the genes indicated that Cel9A consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from N-terminal to C-terminal; Cel12A had only one catalytic domain belonging to glycosyl hydrolase family 12. The combinations of Cel9A and Cel12A produced by the recombinant E. coli exhibited synergistic action against substrates of carboxylmethyl cellulose as well as Avicel.     

Allelic Exchange in Actinomyces oris with mCherry Fluorescence Counterselection

Described here is a method for facile generation of markerless gene deletion mutants of Actinomyces oris. Homologous integration of a nonreplicative vector carrying a gene exchange cassette into the bacterial chromosome was selected for by using mCherry fluorescence and resistance to kanamycin. Completion of allelic replacement was counterselected for by using loss of fluorescence.Actinomyces oris (formerly Actinomyces naeslundii [3]) is Gram positive, facultatively anaerobic, and commonly found in the human oral cavity and plays a major role in the formation of oral biofilm or dental plaque. It is thought that adherence of A. oris to the tooth surface and its coaggregation with oral streptococci create an adhesive platform for subsequent colonization of bacteria in the plaque community (4). A. oris surface molecules such as fimbriae and pili have been shown previously to be required for the bacterial interactions with host tissues and other oral bacteria (7). However, the roles of fimbrial molecules or other surface proteins involved in these processes and their molecular assembly on the cell surface remain elusive. Lack of a facile gene disruption technology is the main reason for this obscurity.Conventional methods of genetic manipulation employing nonreplicative plasmids as delivery vectors in A. oris have been used to create gene disruption by allelic exchange, which allows insertion of a selectable marker (1, 8, 9). Often, this strategy generates polar mutations that affect downstream genes, and it is inadequate for multigene deletion because antibiotic markers for Actinomyces are scarce. To circumvent this problem, we successfully developed a method that utilizes a pUC19 derivative (namely, pHTT177) to generate a nonpolar, in-frame deletion of the sortase gene srtC2 (5). However, this system proved extremely laborious because the second homologous recombination (double-crossover) event leading to chromosomal excision and loss of the plasmid could not be efficiently selected for. Consequently, we explored fluorescence as a positive selection marker for A. oris, as described below.To generate a nonreplicative delivery vector for gene replacement with a counterselectable marker, we cloned the gene encoding the red fluorescent protein mCherry under the control of the constitutive promoter PrpsJ into pHTT177 by using EcoRI and NdeI sites (5) (Fig. ​(Fig.1).1). Initially, the mCherry sequence was amplified from plasmid pRSET-B-mCherry DNA (6) by using primers P1 (5′-GGCGGCTAGCATGGTGAGCAAGGGCGAGGAG-3′) and P2 (5′-GGCGCATATGCTACTACTTGTACAGCTCGTCCATG-3′), which contain NheI and NdeI sites (underlined), respectively. Primers P3 (5′-GGCGGAATTCCGCCCGAGCGCGGGGACCAGT-3′) and P4 (5′-GGCGGCTAGCGGCGCCTAACCTCTCTTGTACTTG-3′), containing EcoRI and NheI sites, were used to amplify the untranslated region of rpsJ from A. oris MG-1 chromosomal DNA (see gene identification no. ANA_0026 in the A. oris database at www.oralgen.lanl.gov). Both fragments were subcloned into pJRD215 at EcoRI and NdeI sites (2). The resulting vector, pCWU3, has a multiple-cloning site (MCS) containing EcoRI, SacI, KpnI, BamHI, XbaI, SalI, and HindIII sites for cloning purposes (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Construction of the nonreplicative delivery vector with red fluorescent mCherry protein as a counterselectable marker. The mCherry gene under the control of the A. oris rpsJ promoter was subcloned into the Escherichia coli/Actinomyces shuttle vector pJRD215 before being cloned into pHTT177, which is a derivative of pUC19. The resulting plasmid, pCWU3, has a kanamycin resistance cassette (kan^R) and an MCS containing EcoRI, SacI, KpnI, BamHI, XbaI, SalI, and HindIII sites.As a proof of concept, we utilized the vector pCWU3, created as described above, to generate an in-frame deletion of acaA (see gene identification no. ANA_0196 in the database at www.oralgen.lanl.gov), encoding a putative cell wall anchor protein (called Aca for actinomyces cell wall anchor). Primer sets P5/P6 (5′-GGCGGAATTCGCCGGAGGCGCCGTCGGGGAAG-3′/5′-GGCGGGTACCAGGATCTCCGTTAGACACGG-3′) and P7/P8 (5′-GGCGGGTACCCAGCGAGACTGCGACCAGCAG-3′/5′-GGCGTCTAGAGGTGGGCGTACTTCTGGTCCAT-3′) were used to amplify ∼1.0-kb sequences upstream and downstream, respectively, of acaA from A. oris MG-1 chromosomal DNA. The upstream DNA fragment was digested with EcoRI and KpnI, while the downstream fragment was digested with KpnI and XbaI (restriction enzyme sites are underlined); both fragments were ligated into pCWU3, which had been precut with EcoRI and XbaI. A. oris MG-1 was transformed with the resulting plasmid by electroporation (5), and kanamycin-resistant colonies representing integration of the plasmid into the bacterial chromosome were selected for their ability to grow on heart infusion (HI; Difco) agar plates supplemented with 50-μg ml⁻¹ kanamycin. These colonies were also examined for their fluorescence by using an Olympus XI71 inverted microscope equipped with a Hamamatsu charge-coupled device camera and a tetramethyl rhodamine isothiocyanate (TRITC) filter set (Fig. ​(Fig.22 A).Open in a separate windowFIG. 2.Analysis of a gene deletion in A. oris. (A) A. oris cells expressing mCherry under the control of the rpsJ promoter were viewed with an Olympus inverted microscope using a TRITC filter. (B and C) Selection of A. oris acaA deletion mutants was performed with a FluorChem Q imaging system (Alpha Innotech, CA). Fluorescent cells appeared green (pseudocolored) with the Cy3 setting, while nonfluorescent cells (potential mutants, indicated by the white arrow in panel C) were gray. An enlarged area (indicated by the white box in panel B) is shown in panel C. (D and E) Nonfluorescent bacteria were further examined for the absence of the acaA gene by PCR amplification (D) and Southern blot analysis (E). Bands of approximately 2.2 kb indicate acaA deletion, whereas bands of approximately 3.3 kb indicate a wild-type (WT) genotype (D). Samples from the parent strain MG-1 (WT) and size markers (M) are indicated, and the black arrow marks a 3.4-kb hybridized fragment.To select Actinomyces clones that had undergone the double-crossover event leading to chromosomal excision and loss of the plasmid, we inoculated a sample of bacteria carrying the integrated plasmid into HI broth overnight at 37°C. The bacterial culture was then serially passaged seven times with a 1:40 dilution in HI broth without antibiotics. Forty-microliter aliquots of the 10,000-fold-diluted final culture were plated onto HI agar plates. After 3 days of growth at 37°C, plates were screened for nonfluorescent colonies by using a FluorChem Q imaging system (Alpha Innotech, CA) with a Cy3 filter. The Cy3 filter was chosen because it produced brighter images than those produced by the Cy5 filter (data not shown) and the images were given with false green coloring (Fig. ​(Fig.2B).2B). Of approximately 16,000 colonies that were screened (a procedure taking less than 30 min), 11 showed no fluorescence (an example indicated by an arrow is shown in Fig. ​Fig.2C),2C), corresponding to a frequency of ∼7.0 × 10⁻⁴. This is consistent with the low frequency of homologous recombination in A. oris (5). Nonfluorescent colonies were also confirmed to be sensitive to kanamycin (data not shown), and their genomic DNA was extracted for PCR and Southern blot analyses. For PCR analysis, primers P5 and P8 were used. As shown in Fig. ​Fig.2D,2D, 8 of the 11 isolates generated amplicons of approximately 2.2 kb, which is indicative of acaA deletion, while the remaining 3 isolates generated the expected wild-type amplicons of approximately 3.3 kb. For further confirmation, DNA samples from three acaA mutants and the wild-type strain MG-1 were analyzed by Southern blotting using a 550-bp probe generated by primers 5′-AGTCTCCAACGCATCCGTCTC-3′ and 5′-GTGTCCCGAGACATTGGCCGTG-3′. Based on sequence analysis of acaA and surrounding genes, digestion by PstI will generate a 3.4-kb fragment for the wild type. As expected, the probe hybridized to the 3.4-kb DNA fragment, which was missing from the three mutant samples (Fig. ​(Fig.2E).2E). Thus, the lack of a hybridization signal for the three mutants further confirmed the absence of acaA in these mutants.In summary, we have developed a facile allelic exchange system for A. oris that reduces the laborious step of screening for a double-crossover event to less than 30 min. To our knowledge, this is the first report of an application that employs a fluorescent protein as a positive selectable marker for gene disruption in bacteria. Conceptually, this strategy can be applied for gene disruption in any system.     

Syntrophomonas wolfei subsp. methylbutyratica subsp. nov., and assignment of Syntrophomonas wolfei subsp. saponavida to Syntrophomonas saponavida sp. nov. comb. nov

A spore-forming bacterium strain 4J5(T) was isolated from rice field mud. When co-cultured with Methanobacterium formicicum DSM 1535(T), strain 4J5(T) could syntrophically degrade saturated fatty acids with 4-8 carbon atoms, including 2-methylbutyrate. Phylogenetic analysis based on 16S rRNA gene similarity showed that strain 4J5(T) was most closely related to Syntrophomonas wolfei subsp. wolfei DSM 2245(T) (98.9% sequence similarity); however, it differed from the latter in the substrates utilized and its genetic characteristics. Therefore, a new subspecies Syntrophomonas wolfei subsp. methylbutyratica is proposed. The type strain is 4J5(T) (=CGMCC 1.5051(T)=JCM 14075(T)). Furthermore, based on 16S rRNA sequence divergence and substrate utilization, we propose the assignment of Syntrophomonas wolfei subsp. saponavida DSM 4212(T) to Syntrophomonas saponavida sp. nov. comb. nov.     

作用于cip-cel mRNA的核糖核酸内切酶的鉴定

【目的】本研究以革兰氏阳性细菌解纤维素梭菌(Ruminiclostridium cellulolyticum)为研究对象,筛选作用于纤维小体编码基因簇cip-cel mRNA的核糖核酸内切酶。【方法】通过对预测的4个假定编码核糖核酸内切酶基因进行基因敲除、体内过表达、体外过表达和活性分析等方法,分析它们对cip-cel mRNA剪切位点的剪切能力。【结果】敲除rnc和rnj基因,对cip-cel mRNA剪切没有任何影响;体内过表达RNase时能够加速cip-cel mRNA的降解,而过表达RNase G时,则结果与野生型对照菌株无异;RNase G基因rng和RNase Y基因rny的体外活性鉴定分析,发现RNase G对体外转录的包含cip-cel mRNA剪切位点的RNA没有作用,而RNase Y能够对其进行剪切和降解。【结论】RNase Y是能够作用于cip-cel mRNA的核酸内切酶。该研究结果对理解革兰氏阳性细菌核糖核酸内切酶的作用机制,及其在转录后水平的调控基因差异表达等方面具有重大意义。     

First Report of Cucumber mosaic virus Infecting Chinese Mallow in China

The virus in naturally infected, stunted Chinese mallow plants and mosaic leaves was identified as Cucumber mosaic virus (CMV). Six symptomatic plants and one symptomless plant were collected in Chongqing, China. DAS‐ELISA suggested CMV was likely associated with the diseased Chinese mallow. Double‐stranded RNA was extracted from the samples, analysed by RT‐PCR, and the coding sequences of their coat proteins (CPs) were sequenced. The results further confirmed CMV was the pathogen causing Chinese mallow stunted, mosaic disease. The isolate was named CMV‐DXC. The full sequence of CMV‐DXC CP was determined, and it had the highest nucleotide identity (99.4%) of those of CMV‐lily, CMV‐WSJ and CMV‐Hnt, respectively. Phylogenetic analysis shows that CMV‐DXC belongs to CMV subgroup II. To our knowledge, this is the first report of CMV infecting Chinese mallow in China.