基于比较基因组学重构细菌的代谢途径和调控网络

微生物基因组学的迅速发展为从功能基因与蛋白、网络及其调控等不同的角度,全面理解与认识微生物的代谢过程、构建细胞工厂,提供了丰富的背景信息。基于基因组序列进行代谢网络重构,有助于发现新的代谢功能基因、调控元件、甚至新的代谢途径,从而优化设计细胞内从原料到产品的生物合成路线。然而,目前公共数据库平台中代谢途径基因的功能注释,许多是错误或不完整的。以下着重介绍了近年来出现的一些用于代谢途径和调控网络重构的新型比较基因组学技术,并以近期的丙酮丁醇梭菌中木糖代谢途径的重构工作为例来说明其应用。     

Evolutionary process of amino acid biosynthesis in Corynebacterium at the whole genome level

Corynebacterium glutamicum, which is the closest relative of Corynebacterium efficiens, is widely used for the large scale production of many kinds of amino acids, particularly glutamic acid and lysine, by fermentation. Corynebacterium diphtheriae, which is well known as a human pathogen, is also closely related to these two species of Corynebacteria, but it lacks such productivity of amino acids. It is an important and interesting question to ask how those closely related bacterial species have undergone such significant functional differentiation in amino acid biosynthesis. The main purpose of the present study is to clarify the evolutionary process of functional differentiation among the three species of Corynebacteria by conducting a comparative analysis of genome sequences. When Mycobacterium and Streptomyces were used as out groups, our comparative study suggested that the common ancestor of Corynebacteria already possessed almost all of the gene sets necessary for amino acid production. However, C. diphtheriae was found to have lost the genes responsible for amino acid production. Moreover, we found that the common ancestor of C. efficiens and C. glutamicum have acquired some of genes responsible for amino acid production by horizontal gene transfer. Thus, we conclude that the evolutionary events of gene loss and horizontal gene transfer must have been responsible for functional differentiation in amino acid biosynthesis of the three species of Corynebacteria.     

Comparative analysis of the primary structure of the reaction center-bound cytochrome subunit in purple bacteria

The amino acid sequences of the reaction center-bound cytochrome subunit of six species of purple bacteria were compared. Amino acid residues thought to be important in controlling the redox midpoint potentials of four hemes in Blastochloris (Rhodopseudomonas) viridis were found to be well conserved. As opposed to all other species studied, the amino acid sequence of the cytochrome subunit of B. viridis had several insertions of more than 10 residues at specific regions close to the LM core, suggesting that interaction of the cytochrome subunit with the LM core in most species is different from that in B. viridis. Distribution of charged amino acid residues on the surface of the cytochrome subunit was compared among six species and discussed from the viewpoint of interaction with soluble electron donors.     

Comprehensive analysis of polyamine transport and biosynthesis in the dominant human gut bacteria: Potential presence of novel polyamine metabolism and transport genes

Recent studies have reported that polyamines in the colonic lumen might affect animal health and these polyamines are thought to be produced by gut bacteria. In the present study, we measured the concentrations of three polyamines (putrescine, spermidine, and spermine) in cells and culture supernatants of 32 dominant human gut bacterial species in their growing and stationary phases. Combining polyamine concentration analysis in culture supernatant and cells with available genomic information showed that novel polyamine biosynthetic proteins and transporters were present in dominant human gut bacteria. Based on these findings, we suggested strategies for optimizing polyamine concentrations in the human colonic lumen via regulation of genes responsible for polyamine biosynthesis and transport in the dominant human gut bacteria.     

醋酸菌中CRISPR位点的比较基因组学与进化分析

CRISPR (Clustered regularly interspaced short palindromic repeats)是近几年发现的一种广泛存在于细菌和古菌中,能够应对外源DNA干扰(噬菌体、病毒、质粒等),并提供免疫机制的重复序列结构。CRISPR系统通常由同向重复序列、前导序列、间隔序列和CRISPR相关蛋白组成。本研究以醋酸发酵中常见3个属醋杆菌属(Acetobacter)、葡糖醋杆菌属(Gluconacetobacter)和葡糖杆菌属(Gluconobacter)的48个菌株为研究对象,通过其基因组上CRISPR相关基因序列的生物信息学分析,探索CRISPR位点在醋酸菌中的多态性及其进化模式。结果表明48株醋酸菌中有32株存在CRISPR结构,大部分CRISPR-Cas结构属于type I-E和type I-C类型。除了葡糖杆菌属外,葡糖醋杆菌属和醋杆菌属中的部分菌株含有II类的CRISPR-Cas系统结构(CRISPR-Cas9)。来自不同属菌株的CRISPR结构中重复序列具有较强的保守性,而且部分菌株CRISPR结构中的前导序列具有保守的motif (与基因的转录调控有关)及启动子序列。进化树分析表明cas1适合用于醋酸菌株的分类,而不同菌株间cas1基因的进化与重复序列的保守性相关,预示它们可能受相似的功能选择压力。此外,间隔序列的数量与噬菌体数量及插入序列(Insertion sequence, IS)数量有正相关的趋势,说明醋酸菌在进化过程中可能正不断受新的外源DNA入侵。醋酸菌中CRISPR结构位点的分析,为进一步研究不同醋酸菌株对醋酸胁迫耐受性差异及其基因组稳定性的分子机制奠定了基础。     

Biosynthesis of ribose-5-phosphate and erythrose-4-phosphate in archaea: a phylogenetic analysis of archaeal genomes

A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP), the ribulose monophosphate (RuMP) pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium) produce ribose-5-phosphate via the nonoxidative PPP (NOPPP), whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1) lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP), the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P) among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii) probably does not synthesize aromatic amino acids at all.     

Comparative sequence analysis of acid sensitive/resistance proteins in Escherichia coli and Shigella flexneri

The molecular basis for the survival of bacteria under extreme conditions in which growth is inhibited is a question of great current interest. A preliminary study was carried out to determine residue pattern conservation among the antiporters of enteric bacteria, responsible for extreme acid sensitivity especially in Escherichia coli and Shigella flexneri. Here we found the molecular evidence that proved the relationship between E. coli and S. flexneri. Multiple sequence alignment of the gadC coded acid sensitive antiporter showed many conserved residue patterns at regular intervals at the N-terminal region. It was observed that as the alignment approaches towards the C-terminal, the number of conserved residues decreases, indicating that the N-terminal region of this protein has much active role when compared to the carboxyl terminal. The motif, FHLVFFLLLGG, is well conserved within the entire gadC coded protein at the amino terminal. The motif is also partially conserved among other antiporters (which are not coded by gadC) but involved in acid sensitive/resistance mechanism. Phylogenetic cluster analysis proves the relationship of Escherichia coli and Shigella flexneri. The gadC coded proteins are converged as a clade and diverged from other antiporters belongs to the amino acid-polyamine-organocation (APC) superfamily.     

Stacking efficiency and flexibility analysis of aromatic amino acids in cap-binding proteins

Recognition of the ribonucleic acid 5' termini (RNA 5' cap) by a wide class of cap-binding proteins is largely accomplished by cation-pi stacking that involves the positively charged 7-methylguanine ring and aromatic amino acid side chains. Quantum calculations of the stacking energy were performed by means of MP2 perturbation method for binary and ternary associates composed of the 7-methylguanine moiety and tryptophan, tyrosine, or phenylalanine, in their spatial orientations known from the crystalline cap-protein complexes. The results clearly pointed to an enhancement of the stacking energy due to a net positive charge in the cap guanine moiety and allowed analysis of a role of various amino acids in stabilization of the complexes. Conformational flexibility of the aromatic amino acids taking part in binding ligands to a wide class of RNA-recognizing proteins, including the cap-binding proteins, was determined by regional order neural network (RONN) algorithm that provides results close to those of the crystallographic B-factors analysis. Interestingly, some of the tyrosines that are classified in general as "rigid" showed high flexibility when engaged in binding the cap to nuclear cap-binding protein complex CBC and to viral methyltransferase VP39. Parallel analyses of the binding energy and flexibility of the protein fragments engaged in the binding leads to understanding differences in molecular mechanisms of the cap recognition by various proteins, CBC compared with the eukaryotic initiation factor eIF4E, and enzymes vs. other protein factors.     

Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis

Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.     

Comparative analysis of conserved genetic markers and adjacent DNA regions identified in beer-spoilage lactic acid bacteria

AIMS: To conduct an inter-species comparative study on the nucleotide sequences of the conserved DNA regions surrounding ORF5, a genetic marker for differentiating beer-spoilage lactic acid bacteria. METHODS AND RESULTS: The conserved DNA regions surrounding ORF5 were examined by PCR analysis, using three beer-spoilage strains, Lactobacillus brevis ABBC45C, L. paracollinoides LA2T and Pediococcus damnosus ABBC478. As a result, the DNA regions containing ORF1-7, originally found in ABBC45C, appeared to be conserved among the three strains, while the downstream region was not found in L. paracollinoides LA2T and P. damnosus ABBC478. The sequencing analysis of the conserved DNA regions of LA2T and ABBC478 revealed ca 99% nucleotide sequence identities with that of ABBC45C. CONCLUSIONS: The nucleotide sequences of the ca 8.2 kb DNA regions containing ORF1-7 were virtually identical among the three strains belonging to different species. The internal organizations of the ORFs were found to be remarkably similar. SIGNIFICANCE AND IMPACT OF THE STUDY: The level of nucleotide sequence identities suggests the DNA regions surrounding ORF5 were horizontally acquired by these beer-spoilage strains belonging to the three different species of lactic acid bacteria.     

副格氏乳杆菌IMAU FB017的遗传背景和益生相关基因分析

【目的】副格氏乳杆菌(Lactobacillus paragasseri)作为格式乳杆菌(Lactobacillus gasseri)的近源物种,是肠道、生殖道重要菌种之一。本研究团队前期发现L. paragasseri IMAU FB017具有良好的胃酸和胆盐耐受性,拟从基因组水平解析IMAU FB017的遗传背景和功能基因特征,并挖掘潜在益生特性基因,为其开发利用奠定遗传学基础。【方法】采用Nanopore和Illumina两种测序技术完成了IMAUFB017全基因组测序及完成图组装,并结合NCBI已公开的18株L.paragasseri基因组序列进行比较基因组学分析。利用Roary软件识别核心基因集与泛基因集;采用Rapid Annotation using Subsystem Technology (RAST)网站对基因组进行功能注释,以探究IMAU FB017基因组特征。【结果】结果显示IMAU FB017基因组包含1条环状染色体和1个质粒,其中环状染色体大小为1 880 023 bp,GC含量为34.90%,包含1 851个蛋白质编码区(protein-coding seq...     

Gene regulatory network from microarray data of colon cancer patients using TSK-type recurrent neural fuzzy network

In this work we applied a TSK-type recurrent neural fuzzy approach to extract regulatory relationship among genes and reconstruct gene regulatory network from microarray data. The identified signature has captured the regulatory relationship among 27 differentially expressed genes from microarray dataset. We applied three different methods viz., feed forward neural fuzzy, modified genetic algorithm and recurrent neural fuzzy, on the same data set for the inference of GRNs and the results obtained are almost comparable. In all tested cases, TRNFN identified more biologically meaningful relations. We found that 87.8% of the total interactions extracted by TRNFN are correct in accordance with the biological knowledge. Our analysis resulted in 2 major outcomes. First, upregulated genes are regulated by more genes than downregulated genes. Second, tumor activators activate other tumor activators and suppress tumor suppressers strongly in the disease environment. These findings will help to elucidate the common molecular mechanism of colon cancer, and provide new insights into cancer diagnostics, prognostics and therapy.     

Compartmental analysis of plasma and liver n-3 essential fatty acids in alcohol-dependent men during withdrawal

The mechanism by which chronic ethanol consumption reduces concentrations of long chain polyunsaturated (LCP) fatty acids (FA) in tissues of humans was investigated in alcohol-dependent (AD) men during early withdrawal and to a well-matched control group by fitting the concentration-time curves of d(5)-labeled n-3 FA from plasma and liver, which originated from an oral dose of d(5)-linolenic acid (d(5)-18:3n-3) ethyl ester to a compartmental model. Blood sampled over 168 h and a liver specimen obtained 96 h after isotope administration were analyzed for d(5)-18:3n-3, d(5)-20:5n-3, d(5)-22:5n-3, and d(5)-22:6n-3. Plasma 20:5n-3 and 22:5n-3 were lower in AD subjects, compared with controls (20:5n-3: -50%, 22:5n-3: -34%). Increased amounts of d(5)-18:3n-3 were directed toward synthesis of d(5)-20:5n-3 in AD subjects (P < .05). However, this effect was offset by larger amounts of 20:5n-3 lost from plasma (control: 2.0 vs. AD: 4.2 mg d(-1)). In livers of AD subjects, more d(5)-18:3n-3 and d(5)-22:5n-3 were utilized for synthesis of d(5)-20:5n-3 (+200%) and d(5)-22:6n-3 (+210%), respectively, than was predicted from plasma kinetics. Although, the potential to utilize linolenic acid for synthesis of LCP FA was greater in AD subjects compared with controls, heightened disappearance rates of 20:5n-3 reduced overall plasma concentrations of several endogenous n-3 LCP FA.     

Evolution of lactic acid bacteria in the order Lactobacillales as depicted by analysis of glycolysis and pentose phosphate pathways

Lactic acid bacteria (LAB) represent a functional group of bacteria that are fundamental in human nutrition because of their prominent role in fermented food production and their presence as commensals in the gut. LAB co-evolution and niche-adaptation have been analyzed in several phylogenomic studies due to the availability of complete genome sequences. The aim of this study was to provide novel insights into LAB evolution through the comparative analysis of the metabolic pathways related to carbohydrate metabolism. The analysis was based on 42 LAB genome sequences of representative strains belonging to Enterococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae. A reference phylogenetic tree was inferred from concatenation of 42 ribosomal proteins; then 42 genes belonging to the Embden–Meyerhof–Parnas (or glycolysis; EMPP) and pentose phosphate (PPP) pathways were analyzed in terms of their distribution and organization in the genomes. Phylogenetic analyses confirmed the paraphyly of the Lactobacillaceae family, while the distribution and organization of the EMPP and PPP genes revealed the occurrence of lineage-specific trends of gene loss/gain within the two metabolic pathways examined. In addition, the investigation of the two pathways as structures resulting from different evolutionary processes provided new information concerning the genetic bases of heterofermentative/homofermentative metabolism.     

Development of multicolour digital image analysis system to enumerate actively respiring bacteria in natural river water

AIMS: To develop a rapid and simple method for the assessment of metabolic activity of bacteria in natural environment. METHODS AND RESULTS: A rapid and simple multicolour digital image analysis system for enumerating viable bacteria based on active fluorescent staining has been developed. This system can accurately differentiate actively respiring bacteria and non-respiring bacteria by distinctive colour information in the digital image captured by an epifluorescence microscope equipped with low magnification objective lens. An algorithm to distinguish bacteria from considerable detritus, which produced bright fluorescence in different colours, by colour segmentation has also been developed. This system was applied to river water samples, and the total and respiratory active bacterial counts by digital image analysis were highly related to those by epifluorescence microscopy (r2 = 0.96 and 0.93, respectively). CONCLUSION: This system allowed the rapid and simple differentiation of bacteria from detritus and concurrent assessment of their metabolic activity, with results being available within 1 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The low magnification image analysis allows more rapid and simple quantification of bacteria and could considerably decrease image data amount and acquisition time. This system could be easily applied to the rapid analysis of microbes in various environments.     

Comparative analysis of caveolins in mouse and tammar wallaby: Role in regulating mammary gland function

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70–80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.