Inter- and intra-genomic heterogeneity of the intervening sequence in the 23S ribosomal RNA gene of Campylobacter jejuni and Campylobacter coli

An intervening sequence (IVS) can be present or absent in the 23S rRNA of Campylobacter jejuni and Campylobacter coli. As part of a survey, we used a polymerase chain reaction (PCR) assay to detect the presence of the IVS in 43 isolates of C. coli and 82 isolates of C. jejuni. An IVS was present in 40 (93.0%) of the C. coli and only 34 (41.5%) of the C. jejuni isolates. Twelve (27.9%) of the C. coli isolates and seven (8.5%) of the C. jejuni isolates resulted in two polymerase chain reaction products, indicating heterogeneity in the presence of the 23S rRNA IVS. Fourteen of the isolates with two products were evaluated by pulse-field gel electrophoresis; 13 different patterns were observed. The total band size of one isolate was substantially greater than the expected 1.7 Mb, possibly indicating a mixed culture. Southern blot analyses demonstrated the expected three rRNA operons in all tested isolates. Nested PCR reactions with operon-specific primers followed by primers for the IVS confirmed that the strains of interest contained either one or two operons carrying the IVS and the remaining operon(s) did not. Sequence analysis of the IVS and flanking regions of the 23S rRNA genes did not discriminate C. jejuni and C. coli as distinct populations. These results indicate horizontal transfer of 23S rRNA genes or portions of the genes between C. jejuni and C. coli. Also, data showing sequence polymorphisms between the three 23S rRNA loci outside of the IVS region suggest that the isolates with intra-genomic heterogeneity appear to be members of clones that have an ancient defect in gene conversion mechanisms needed for concerted evolution of the ribosomal operons.     

Comet Assay measurements: a perspective

The Comet Assay or single cell gel electrophoresis assay is one of the very widely used assays to microscopically detect DNA damage at the level of a single cell. The determination of damage is carried out either through visual scoring of cells (after classification into different categories on the basis of tail length and shape) or by using different commercially available or public domain software (which automatically recognise the extent of damage). In this assay, the shape, size and amount of DNA within the ‘comet’ play important roles in the determination of the level of damage. The use of a software in particular also provides a range of different parameters, many of which might not be relevant in determining the extent of DNA damage. As a large number of factors could influence the shape, size, identification and determination of induced damage, which includes the scoring criteria, staining techniques, selection of parameters (whilst using the software packages) and appearance of ‘hedgehog’ or ‘clouds’, this article aims (a) to provide an overview of evolution of measurements of DNA damage using the Comet Assay and (b) to summarise and critically analyse the advantages and disadvantages of different approaches currently being adopted whilst using this assay. It is suggested that judicious selection of different parameters, staining methods along with inter-laboratory validation and harmonisation of methodologies will further help in making this assay more robust and widely acceptable for scientific as well as regulatory studies.     

Development of a generic approach to native metalloproteomics: application to the quantitative identification of soluble copper proteins in Escherichia coli

A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁷W, ⁹⁹Mo, ⁶⁹Zn, ⁵⁶Mn, ⁶⁵Ni); (2) separation of intact soluble holoproteins using native isoelectric focusing combined with blue native polyacrylamide gel electrophoresis into native–native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. The method was applied to the identification of copper proteins from a soluble protein extract of wild-type Escherichia coli K12 using the radioisotope ⁶⁴Cu. The E. coli protein CueO, which has previously been only identified as a multicopper oxidase following homologous overexpression, was now directly detected as a copper protein against a wild-type background at an expression level of 0.007% of total soluble protein. The retention of the radioisotope by the copper proteins throughout the separation process corroborates the method to be genuinely native. The procedure developed here can be applied to cells of any origin, and to any metal having suitable radioisotopes. The finding that the periplasmic protein CueO is the only major form of soluble protein bound copper in E. coli strengthens the view that the bacterial periplasm contains only a few periplasmic copper proteins, and that the cytosol is devoid of copper proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The genetic consequence of differing ecological demands of a generalist and a specialist butterfly species

Many studies aim at testing the impact of recent fragmentation on the genetic diversity and connectivity of populations, while some species do exist naturally in fragmented landscapes because of their habitat requirements. Therefore, it is important to look at the genetic signatures of species occurring in naturally fragmented landscapes in order to disentangle the effect of fragmentation from the effect of habitat requirements. We selected two Nymphalid butterflies for this purpose. While Melanargia galathea is a common butterfly in flower-rich meadows, Melitaea aurelia is closely connected to calcareous grasslands, thus being historically fragmented due to its ecological demands. For the analysis of the genetic response on these opposed patterns, we analysed 18 allozyme loci for 789 individuals (399 individuals of M. galathea and 390 individuals of M. aurelia) in a western German study region with adjacent areas in Luxemburg and northeastern France. Both species showed similarly low genetic differentiations among local populations (M. galathea: F _ST 3.3%; M. aurelia: F _ST 3.6%), both combined with a moderate level of inbreeding. Isolation-by-distance analysis revealed a significant correlation for both species with similar amounts of explained variances (M. galathea: r ² = 27.8%; M. aurelia: r ² = 28.5%). Most parameters of genetic diversity were higher in M. galathea than in M. aurelia, but the latter species had a considerably higher amount of rare or locally restricted genes; the differing ecological demands are thus reflected in these differences. Both species thus seem to be genetically well suited to their respective ecological requirements. In the light of conservation genetics, we deduce that highly fragmented populations are not necessarily prone to extinction. The extinction risk might be linked to the life history of an organism and its population genetic structure.     

Optimized isolation procedure for obtaining strongly actinide binding exopolymeric substances (EPS) from two bacteria (Sagittula stellata and Pseudomonas fluorescens Biovar II)

Different chemical extractants (NaCl, EDTA, HCl and NaOH) and physical methods (ultrasonication and heating) were examined by their efficacies of extracting “attached” exopolymeric substances (EPS) secreted by marine bacterium Sagittula stellata (SS) and terrestrial bacterium Pseudomonas fluorescens Biovar II (PF). Extraction by 0.5 N HCl for 3 h was best for SS while extraction by 0.05 N NaCl for 3–5 h was regarded as optimal for PF. Improvements in EPS purification included a pre-diafiltration step to remove the broth material and reduce the solution volume, thus the usage of ethanol, and time. The EPS harvested at the optimal time and purified by the improved method were enriched in polysaccharides, with smaller amounts of proteins, thus having amphiphilic properties. Isoelectric focusing of ²³⁴Th or ²⁴⁰Pu labeled EPS showed both actinides were strongly bound to macromolecules with low pI, similar to reported marine or soil colloidal natural organic matter (NOM).     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.     

121株甲型副伤寒病原菌的噬菌体型和脉冲场凝胶电泳型

目的认识甲型副伤寒疫病区甲型副伤寒沙门菌的噬菌体型和脉冲场凝胶电泳（PFGE）型,确定噬菌体型和PFGE型之间的关系以及菌型的分布和流行率。方法采用沙门菌组合噬菌体和SpeI、XbaI消化染色体DNA的PFGE对来自玉溪市7县（区）的121株甲型副伤寒菌进行分型。结果121株菌存在4个完全噬菌体型或1个噬菌体型;用SpeI或XbaI消化产物分别得出以SpeI01、SpeI02或XbaI01占优势的5种或4种PFGE型,SpeI01型和SpeI02型分别占37．2％和57．9％,XbaI01型占95．1％。结论121株菌的噬菌体型与PFGE型之间无一致性联系,PFGE型的SpeI01和SpeI02或XbaI01是玉溪地区的主要流行型,采用SpeI和XbaI的PFGE是鉴别甲型副伤寒菌流行克隆的一项有用技术。     

几种动物肝脏糖胺聚糖的醋酸纤维素薄膜电泳分析

采用酶解和离子交换色谱的方法,从兔、鸡、猪和羊肝组织中提取和纯化得到了糖胺聚糖（GAGs）.通过比较透明质酸（HA）、硫酸软骨素A（CS-A）、硫酸软骨素C（CS-C）、硫酸皮肤素（DS）、肝素（HP）、硫酸乙酰肝素（HS）等标准品在醋酸钡、醋酸锌、吡啶-甲酸等几种不同缓冲体系下的醋酸纤维素薄膜电泳行为,结合灰度积分建立了适合于微量GAGs定性和定量分析的电泳方法.将从不同动物肝脏组织中提取的GAGs运用该方法进行分析,发现 不同动物肝脏组织中,GAG含量和组成均有较大差异：羊肝中GAGs含量最高（0.52 mg/g 组织干粉）,种类也最丰富,含有HA、HS、DS和CS,其中HA所占比例最高;鸡肝中GAGs含量最少（0.18 mg/g组织干粉）,主要含有HA和DS;兔肝GAGs种类与猪肝相似,均含有HA、HS和DS,但HS是猪肝GAGs的主要成分,DS是兔肝GAGs的主要成分.     

毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.     

Bacterial 'cosmopolitanism' and importance of local environmental factors for community composition in remote high-altitude lakes

1. In October 2004, plankton samples were collected from six permanent lakes located between 4960 and 5440 m a.s.l. in the Mount Everest region (Nepal) to assess how spatial and local environmental factors affect natural bacterial community composition. Fingerprinting analysis of the bacterial 16S rRNA gene fragment was done by denaturing gradient gel electrophoresis (DGGE).
2. The number of DGGE bands (range: 12–23) was not correlated with lake area or remoteness, but there was a strong negative correlation with the ratio of catchment to lake area ( r  = −0.826, P  <   0.05), suggesting that hydraulic retention time affects the establishment of the bacterial community in these seepage lakes.
3. Most dominant sequences belonged to Betaproteobacteria except in two lakes where members of Bacteroidetes made the largest relative contribution. Up to 81% of the phylotypes had high similarity (>98 to 100%) in partial 16S rRNA gene sequence to those reported from other alpine lakes and glaciers around the world, suggesting the presence of 'cosmopolitan' bacteria.
4. An analysis based on dissimilarity matrices and the Mantel test revealed the existence of dissimilarities in bacterial community composition related to geographical distance over a small spatial scale (<6 km), but determined by local environmental constraints.
5. Our results suggest that several bacterial phylotypes are ubiquitous in the freshwater aquatic realm, but taxon sorting by local environmental constraints is important.