Features for computational operon prediction in prokaryotes

Accurate prediction of operons can improve the functional annotation and application of genes within operons in prokaryotes. Here, we review several features: (i) intergenic distance, (ii) metabolic pathways, (iii) homologous genes, (iv) promoters and terminators, (v) gene order conservation, (vi) microarray, (vii) clusters of orthologous groups, (viii) gene length ratio, (ix) phylogenetic profiles, (x) operon length/size and (xi) STRING database scores, as well as some other features, which have been applied in recent operon prediction methods in prokaryotes in the literature. Based on a comparison of the prediction performances of these features, we conclude that other, as yet undiscovered features, or feature selection with a receiver operating characteristic analysis before algorithm processing can improve operon prediction in prokaryotes.     

Genome analysis for nucleotide interactions in fully sequenced genomes of selective prokaryotes

Fully sequenced prokaryotic genomes ofEscherichia coli, Haemophilus influenzae andMethanococcus janaschii were subjected .to genome analysis for nucleotide interactions. The analysis was restricted to inter-nucleotide relations like two nucleotides in a dinucleotide, three nucleotides in a codon and two codons in a dicon. This relational analysis was carried out in C language and was compiled on a C++ compiler. The relational analysis showed a preferential dinucleotide frequency (the observed frequencies of AA and TT were higher than the expected frequency and the observed frequencies of CC and GG). From codon frequency distribution analysis, sub-codonic elements have been noticed, exerting that the first one or first two nucleotide may reasonably determine the next nucleotide(s) in a codon. The analysis further reveals the existence of short-range randomness or chaotic behaviour in prokaryotic genomes, which might be a forerunner for the origin of introns in eukaryotes, besides being involved in a regulatory role.     

A novel class of modular transporters for vitamins in prokaryotes

The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters.     

Assignment of isochores for all completely sequenced vertebrate genomes using a consensus

We show that although the currently available isochore mapping methods agree on the isochore classification of about two-thirds of the human DNA, they produce significantly different results with regard to the location of isochore boundaries and isochore length distribution. We present a new consensus isochore assignment method based on majority voting and provide IsoBase, a comprehensive on-line database of isochore maps for all completely sequenced vertebrate genomes.     

Comparative genomics analysis of completely sequenced microbial genomes reveals the ubiquity of N-linked glycosylation in prokaryotes

Glycosylation of proteins in prokaryotes has been known for the last few decades. Glycan structures and/or the glycosylation pathways have been experimentally characterized in only a small number of prokaryotes. Even this has become possible only during the last decade or so, primarily due to technological and methodological developments. Glycosylated proteins are diverse in their function and localization. Glycosylation has been shown to be associated with a wide range of biological phenomena. Characterization of the various types of glycans and the glycosylation machinery is critical to understand such processes. Such studies can help in the identification of novel targets for designing drugs, diagnostics, and engineering of therapeutic proteins. In view of this, the experimentally characterized pgl system of Campylobacter jejuni, responsible for N-linked glycosylation, has been used in this study to identify glycosylation loci in 865 prokaryotes whose genomes have been completely sequenced. Results from the present study show that only a small number of organisms have homologs for all the pgl enzymes and a few others have homologs for none of the pgl enzymes. Most of the organisms have homologs for only a subset of the pgl enzymes. There is no specific pattern for the presence or absence of pgl homologs vis-à-vis the 16S rRNA sequence-based phylogenetic tree. This may be due to differences in the glycan structures, high sequence divergence, horizontal gene transfer or non-orthologous gene displacement. Overall, the presence of homologs for pgl enzymes in a large number of organisms irrespective of their habitat, pathogenicity, energy generation mechanism, etc., hints towards the ubiquity of N-linked glycosylation in prokaryotes.     

A novel dnaK operon from Porphyromonas gingivalis

The nucleotide sequence of the dnaK operon cloned from Porphyromonas gingivalis revealed that the operon does not contain homologues of either dnaJ or grpE. However, there were two genes which encode small heat shock proteins immediately downstream from the dnaK and they were transcribed together with dnaK as one unit. The ATPase activity of the P. gingivalis DnaK was synergistically stimulated up to 40-fold in the simultaneous presence of Escherichia coli DnaJ and GrpE. These results suggest that the DnaK homologue of P. gingivalis, with its unique genetic structure and evolutionary features, works as a member of the DnaK chaperone system.     

A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes

The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications.     

A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids

Administered subcutaneously, D-4F or L-4F are equally efficacious, but only D-4F is orally efficacious because of digestion of L-4F by gut proteases. Orally administering niclosamide (a chlorinated salicylanilide used as a molluscicide, antihelminthic, and lampricide) in temporal proximity to oral L-4F (but not niclosamide alone) in apoE null mice resulted in significant improvement (P < 0.001) in the HDL-inflammatory index (HII), which measures the ability of HDL to inhibit LDL-induced monocyte chemotactic activity in endothelial cell cultures. Oral administration of L-[113-122]apoJ with niclosamide also resulted in significant improvement (P < 0.001) in HII. Oral administration of niclosamide and L-4F together with pravastatin to female apoE null mice at 9.5 months of age for six months significantly reduced aortic sinus lesion area (P = 0.02), en face lesion area (P = 0.033), and macrophage lesion area (P = 0.02) compared with pretreatment, indicating lesion regression. In contrast, lesions were significantly larger in mice receiving only niclosamide and pravastatin or L-4F and pravastatin (P < 0.001). In vitro niclosamide and L-4F tightly associated rendering the peptide resistant to trypsin digestion. Niclosamide itself did not inhibit trypsin activity. The combination of niclosamide with apolipoprotein mimetic peptides appears to be a promising method for oral delivery of these peptides.     

A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea

Multicellular magnetotactic prokaryotes (MMPs) are a group of magnetotactic microorganisms composed of 10-40 Gram-negative cells. Currently, all the identified MMPs show a spherical morphology and synthesize mainly iron sulfide magnetosomes. In this study, we report a novel genus of MMPs with peculiar ellipsoidal morphology and iron oxide magnetosomes, which were discovered in intertidal sediment of the Yellow Sea in China. Optical and fluorescence microscopy revealed that this organism was ~10 × 8 μm in size and composed of ~40 cells enveloped by an outer layer. Scanning electron microscopy showed that the cells were arranged in 4-6 interlaced circles. Bullet-shaped magnetite magnetosomes were organized in chains roughly parallel to the long axis of the ellipsoidal MMPs when analysed by transmission electron microscopy. These MMPs displayed special escape motility, i.e. swimming rapidly from the edge to the centre of the droplet and then slowly back to the edge. In addition, they exhibited negative phototaxis. Light microscopy observations showed that the ellipsoidal MMPs reproduced by division along the body long axis. Both analysis of 16S rRNA gene sequence and fluorescence in situ hybridization revealed the ellipsoidal MMPs as a new genus of the Deltaproteobacteria. In summary, this novel genus of MMPs exhibit unique morphology, peculiar division process and distinct phylogenetic affiliation compared with the other MMPs.     

A natural species concept for prokaryotes

Direct molecular analyses of natural microbial populations reveal patterns that should compel microbiologists to adopt a more natural species concept that has been known to biologists for decades. The species debate can be exploited to address a larger issue - microbiologists need, in general, to take a more natural view of the organisms they study.     

A simple method for accurate estimation of apoptotic cells

A simple, sensitive, and reliable "DNA diffusion" assay for the quantification of apoptosis is described. Human lymphocytes and human lymphoblastoid cells, MOLT-4, were exposed to 0, 12.5, 25, 50, or 100 rad of X-rays. After 24 h of incubation, cells were mixed with agarose, microgels were made, and cells were lysed in high salt and detergents. DNA was precipitated in microgels by ethanol. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a halo of granular DNA with a hazy outer boundary. Necrotic cells, resulting from hyperthermia treatment, on the other hand, show an unusually large homogeneous nucleus with a clearly defined boundary. The number of cells with apoptotic and necrotic appearance can be scored and quantified by using a fluorescent microscope. Results were compared with other methods of apoptosis measurement: morphological estimations of apoptosis and DNA ladder pattern formation in regular agarose gel electrophoresis. Validation of the technique was done using some known inducers of apoptosis and necrosis (hyperthermia, hydrogen peroxide, mitoxantrone, novobiocin, and sodium ascorbate).     

一种快速精确编辑疱疹病毒基因组的方法

为了快速且准确地对疱疹病毒基因组进行基因敲除、插入或者点突变等修饰,通过同源重组将马立克氏病病毒 (MDV) 超强毒株Md5基因组克隆到细菌人工染色体 (BAC)。将筛选的阳性重组体DNA电转进DH10B菌株,用PCR及限制性片段多态分析 (RFLP) 方法鉴定含Md5全基因组的BAC克隆。将阳性重组体DNA转染入鸡胚成纤维细胞 (CEF),拯救出重组病毒,命名为Md5BAC。进一步利用Red酶介导的两步法基因重组技术构建MDVlorf10基因敲除毒株。为了验证被敲除基因功能的特异性,将lorf10插入原位点以构建基因复原毒株。将构建的重组毒株分别感染CEF细胞,用间接免疫荧光试验确认重组病毒均包装成功;病毒生长曲线结果表明,lorf10敲除不影响病毒的体外增殖。总之,这为其他疱疹病毒的基因组编辑提供了技术参考。     

A new type of bacterial sulfatase reveals a novel maturation pathway in prokaryotes

Sulfatases are a highly conserved family of enzymes found in all three domains of life. To be active, sulfatases undergo a unique post-translational modification leading to the conversion of either a critical cysteine ("Cys-type" sulfatases) or a serine ("Ser-type" sulfatases) into a Calpha-formylglycine (FGly). This conversion depends on a strictly conserved sequence called "sulfatase signature" (C/S)XPXR. In a search for new enzymes from the human microbiota, we identified the first sulfatase from Firmicutes. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that this enzyme undergoes conversion of its critical cysteine residue into FGly, even though it has a modified (C/S)XAXR sulfatase signature. Examination of the bacterial and archaeal genomes sequenced to date has identified many genes bearing this new motif, suggesting that the definition of the sulfatase signature should be expanded. Furthermore, we have also identified a new Cys-type sulfatase-maturating enzyme that catalyzes the conversion of cysteine into FGly, in anaerobic conditions, whereas the only enzyme reported so far to be able to catalyze this reaction is oxygen-dependent. The new enzyme belongs to the radical S-adenosyl-l-methionine enzyme superfamily and is related to the Ser-type sulfatase-maturating enzymes. This finding leads to the definition of a new enzyme family of sulfatase-maturating enzymes that we have named anSME (anaerobic sulfatase-maturating enzyme). This family includes enzymes able to maturate Cys-type as well as Ser-type sulfatases in anaerobic conditions. In conclusion, our results lead to a new scheme for the biochemistry of sulfatases maturation and suggest that the number of genes and bacterial species encoding sulfatase enzymes is currently underestimated.     

A novel species of ellipsoidal multicellular magnetotactic prokaryotes from Lake Yuehu in China

Two morphotypes of multicellular magnetotactic prokaryotes (MMPs) have been identified: spherical (several species) and ellipsoidal (previously one species). Here, we report novel ellipsoidal MMPs that are ～ 10 × 8 μm in size, and composed of about 86 cells arranged in six to eight interlaced circles. Each MMP was composed of cells that synthesized either bullet‐shaped magnetite magnetosomes alone, or both bullet‐shaped magnetite and rectangular greigite magnetosomes. They showed north‐seeking magnetotaxis, ping‐pong motility and negative phototaxis at a velocity up to 300 μm s^?1. During reproduction, they divided along either their long‐ or short‐body axes. For genetic analysis, we sorted the ellipsoidal MMPs with micromanipulation and amplified their genomes using multiple displacement amplification. We sequenced the 16S rRNA gene and found 6.9% sequence divergence from that of ellipsoidal MMPs, Candidatus Magnetananas tsingtaoensis and > 8.3% divergence from those of spherical MMPs. Therefore, the novel MMPs belong to different species and genus compared with the currently known ellipsoidal and spherical MMPs respectively. The novel MMPs display a morphological cell differentiation, implying a potential division of labour. These findings provide new insights into the diversity of MMPs in general, and contribute to our understanding of the evolution of multicellularity among prokaryotes.     

A qRT-PCR-based method for the measurement of rrn operon copy number

Aim:  To develop a convenient and accurate method for estimating the rrn operon copy number ( Y _rrn ) in cells of pure prokaryotic cultures based on quantitative real-time polymerase chain reaction (qRT-PCR).
Methods & Results:  Using Escherichia coli, the Y _rrn of which is known to be 7, as a reference, the rrn concentrations of target species and E. coli in sample solutions were measured based on their respective threshold cycle numbers ( C _t ), whereas the cell concentrations of both species were measured by microscopic counting after staining. The Y _rrn of the target species was then calculated from the initial cell concentrations and the rrn concentrations of the target species and E. coli . Using this method, the Y _rrn values of four species, i.e. Xanthomonas campestris , Staphylococcus aureus , Aeromonas hydrophila and Pseudomonas fluorescens , were estimated as 1·80, 4·73, 8·58 and 5·13, respectively, comparable to their respective known values of 2, 5, 10, and 5, resulting in an average deviation of 8%.
Conclusions:  The whole cell qRT-PCR based methods were convenient, accurate and reproducible in quantification of rrn copy number of prokaryotic cells.
Significance and Impact of the Study:  qTR-PCR is a fast and reliable DNA quantification approach. Compared with previous qTR-PCR based methods measuring rrn copy number, the present method avoided the prerequisite for the information on genome size and GC content of target bacteria or a gene with known copy number, thus should be more widely applicable.     

iPcc: a novel feature extraction method for accurate disease class discovery and prediction

Gene expression profiling has gradually become a routine procedure for disease diagnosis and classification. In the past decade, many computational methods have been proposed, resulting in great improvements on various levels, including feature selection and algorithms for classification and clustering. In this study, we present iPcc, a novel method from the feature extraction perspective to further propel gene expression profiling technologies from bench to bedside. We define ‘correlation feature space’ for samples based on the gene expression profiles by iterative employment of Pearson’s correlation coefficient. Numerical experiments on both simulated and real gene expression data sets demonstrate that iPcc can greatly highlight the latent patterns underlying noisy gene expression data and thus greatly improve the robustness and accuracy of the algorithms currently available for disease diagnosis and classification based on gene expression profiles.