Replacement of the Arginine Biosynthesis Operon in Xanthomonadales by Lateral Gene Transfer

The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the γ-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.     

细菌胞外多糖的生物合成与基因控制

细菌胞外多糖是指细菌在生长发育过程中合成并分泌到细胞外的长链,高分子糖类聚合物。细菌胞外多糖的生物合成途径涉及装配、多聚化及运输三个过程,是多种酶和转运系统的结果,其发生的部位包括胞内和胞外,有些合成过程会发生在细胞壁上,对于胞外多糖合成相关基因的报道,发现控制胞外多糖合成是一大类基因簇,不同的菌株其基因簇的数量和种类各不相同。这些研究的不断更新为将来胞外多糖的应用提供了更加广阔的前景。     

变构菌素生物合成基因簇的研究进展

变构菌素是从Streptomyces griseochromogenes菌株中分离得到的第一个高选择性的蛋白磷酸化酶-1（PP-1）抑制剂。变构菌素及其衍生物在神经系统紊乱、代谢综合症、呼吸系统及相关疾病、免疫抑制、肿瘤治疗等诸多领域都有着广泛的应用前景,因而引起了人们对其生物合成途径的研究兴趣。介绍了近年来变构菌素生物合成途径的研究进展。     

蓝藻基因转移系统的选择与建立

蓝藻是一类进行光合放氧的原核生物 ,因其结构的特殊性 ,已成为表达外源基因的理想宿主之一。然而外源基因转化系统的选择与建立一直影响着蓝藻基因工程的快速发展。总结了各类蓝藻基因转移系统的特点、影响因素、各系统间的优缺点、以及不同蓝藻株系最适基因转移系统的选择等 ,为利用蓝藻进行遗传操作提供可能 ,为蓝藻基因工程发展提供信息。     

Evidence for Multiple Lateral Transfers of the Circadian Clock Cluster in Filamentous Heterocystic Cyanobacteria Nostocaceae

Cyanobacteria are the first prokaryotes reported to show circadian rhythmicity, which is regulated by a cluster of three genes: kaiA, kaiB, and kaiC. Phylogenetic analysis of the kaiBC cluster in filamentous cyanobacteria of the family Nostocaceae including Nodularia spumigena and Nostoc linckia from Arubotaim Cave, Mt. Sedom, Israel, indicated that this cluster has experienced multiple lateral transfers. The transfers have occurred in different periods of the species evolution. The data obtained suggest that lateral transfers of the circadian clock cluster in filamentous cyanobacteria have been common and might have adaptive significance.     

Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis

Toxic cyanobacterial blooms cause economic losses and pose significant public health threats on a global scale. Characterization of the gene cluster for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 is described, and the complete biosynthetic pathway is proposed. The cyr gene cluster spans 43 kb and is comprised of 15 open reading frames containing genes required for the biosynthesis, regulation, and export of the toxin. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions and subsequent reductions, and rings are formed via Michael additions in a stepwise manner. The uracil ring is formed by a novel pyrimidine biosynthesis mechanism and tailoring reactions, including sulfation and hydroxylation that complete biosynthesis. These findings enable the design of toxic strain-specific probes and allow the future study of the regulation and biological role of cylindrospermopsin.     

系统发育分析指示细菌向Apicoplast的水平基因转移

顶复合器门的原生动物(Apicomplexan protozoa)含有一个高度退化的质体样(plastid-like)细胞器,定名为apicoplast。来自apicoplast的c／pC基因和它在其他质体和细菌中的同源物用来重建apicoplast的系统发育史。使用邻接法(Neighbor-Joining)、最小进化法(Minimum Evolution)、最大简约法(Maximum Pamimony)和最大似然法(Maximum Likelihood)建立进化树。此外为了避免由于序列之间相似的碱基组成而引起的虚假聚类,建立了基于LogDet距离的核苷酸NJ树;以及为了避免由于在核苷酸和氨基酸水平上的突变饱和而引起的长分枝吸引(Long Branch Attraction,LBA),建立了基于非饱和位点的核苷酸和氨基酸序列的系统发育树。重建结果强有力地支持apicoplast和细菌B．buigorferi之间的单系(monophyly)起源关系,也强化了apicoplasst属于混合基因组的假设,并且提供了对这个混合基因组起源的新的认识。     

Characterization of an Ancient Lepidopteran Lateral Gene Transfer

Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea). Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material.     

噬藻体和蓝藻间的基因转移及协同进化作用

生物物种之间的水平基因转移广泛存在于细菌、古生菌和真核生物中,并能造成同一生境中种群的快速协同进化。噬藻体是感染蓝藻的专一性病毒,近年研究表明其在蓝藻水华生消中发挥了重要作用,使人们认识到了噬藻体的重要生态地位。综述了物种间的水平基因转移,介绍了噬藻体遗传多样性研究中常用的光合作用基因、结构蛋白基因等靶标基因所介导的基因转移以及基因转移引起的病毒和宿主的协同进化,并介绍了研究基因转移所用到的试验技术以及今后所要面临的问题。     

Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis

Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans.     

Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii

Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.     

Citrullination Was Introduced into Animals by Horizontal Gene Transfer from Cyanobacteria

Protein posttranslational modifications add great sophistication to biological systems. Citrullination, a key regulatory mechanism in human physiology and pathophysiology, is enigmatic from an evolutionary perspective. Although the citrullinating enzymes peptidylarginine deiminases (PADIs) are ubiquitous across vertebrates, they are absent from yeast, worms, and flies. Based on this distribution PADIs were proposed to have been horizontally transferred, but this has been contested. Here, we map the evolutionary trajectory of PADIs into the animal lineage. We present strong phylogenetic support for a clade encompassing animal and cyanobacterial PADIs that excludes fungal and other bacterial homologs. The animal and cyanobacterial PADI proteins share functionally relevant primary and tertiary synapomorphic sequences that are distinct from a second PADI type present in fungi and actinobacteria. Molecular clock calculations and sequence divergence analyses using the fossil record estimate the last common ancestor of the cyanobacterial and animal PADIs to be less than 1 billion years old. Additionally, under an assumption of vertical descent, PADI sequence change during this evolutionary time frame is anachronistically low, even when compared with products of likely endosymbiont gene transfer, mitochondrial proteins, and some of the most highly conserved sequences in life. The consilience of evidence indicates that PADIs were introduced from cyanobacteria into animals by horizontal gene transfer (HGT). The ancestral cyanobacterial PADI is enzymatically active and can citrullinate eukaryotic proteins, suggesting that the PADI HGT event introduced a new catalytic capability into the regulatory repertoire of animals. This study reveals the unusual evolution of a pleiotropic protein modification.     

Characterization of the Complete Zwittermicin A Biosynthesis Gene Cluster from Bacillus cereus

Bacillus cereus UW85 produces the linear aminopolyol antibiotic zwittermicin A (ZmA). This antibiotic has diverse biological activities, such as suppression of disease in plants caused by protists, inhibition of fungal and bacterial growth, and amplification of the insecticidal activity of the toxin protein from Bacillus thuringiensis. ZmA has an unusual chemical structure that includes a d amino acid and ethanolamine and glycolyl moieties, as well as having an unusual terminal amide that is generated from the modification of the nonproteinogenic amino acid β-ureidoalanine. The diverse biological activities and unusual structure of ZmA have stimulated our efforts to understand how this antibiotic is biosynthesized. Here, we present the identification of the complete ZmA biosynthesis gene cluster from B. cereus UW85. A nearly identical gene cluster is identified on a plasmid from B. cereus AH1134, and we show that this strain is also capable of producing ZmA. Bioinformatics and biochemical analyses of the ZmA biosynthesis enzymes strongly suggest that ZmA is initially biosynthesized as part of a larger metabolite that is processed twice, resulting in the formation of ZmA and two additional metabolites. Additionally, we propose that the biosynthesis gene cluster for the production of the amino sugar kanosamine is contained within the ZmA biosynthesis gene cluster in B. cereus UW85.Bacillus cereus strain UW85 was isolated for its ability to suppress disease in alfalfa caused by the plant pathogen Phytophthora medicaginis (17). This antiprotist activity was subsequently found to be associated with the filtrate of fully sporulated B. cereus UW85 (37). Analysis of this filtrate identified two antiprotist antibiotics, zwittermicin A (ZmA) and kanosamine (28, 37). Of the two antibiotics, ZmA has shown the more interesting biological activities, having not only antiprotist activity, but also antibiotic activity against gram-positive and gram-negative bacteria, as well as fungi (32, 38). ZmA was also found to potentiate the activity of the toxin protein of Bacillus thuringiensis against insects (3).A preliminary chemical structure of ZmA was determined by the Handelsman and Clardy groups (18). More recently, Rogers and colleagues performed a series of elegant structural studies that compared ZmA produced from B. cereus with synthetic ZmA derivatives that had varied stereocenters (32, 33). From this work, the chemical structure of ZmA with the appropriate stereocenters has been determined (Fig. ​(Fig.1).1). The antibiotic has a number of unusual structural components. First, ZmA is one of only a few linear aminopolyol natural products to be identified. Second, the core of ZmA is formed from ethanolamine and glycolyl moieties that are rarely seen in natural products. Third, the N terminus of ZmA is formed from d-serine (d-Ser), not l-Ser, as initially expected. This suggests that the amino acid either is incorporated as the d isomer or is incorporated as the l isomer and is then isomerized at some point during its biosynthesis. Finally, ZmA is the only natural product that we are aware of that contains an unusual 2-aminosuccinamide moiety. This moiety is likely to come from the amino acid β-ureidoalanine (β-Uda) that has had its carboxylic acid replaced by a terminal amide.Open in a separate windowFIG. 1.Chemical structure of ZmA. Numbers have been added to identify the sites of hydroxyl groups as discussed in the text.We have been investigating how B. cereus UW85 assembles this antibiotic to gain insights into how production of ZmA can be improved and how the unusual structural components of ZmA are formed. We previously proposed that the biosynthesis of ZmA involves the condensation of the amino acids l-Ser and l-2,3-diaminopropionate (l-Dap), along with the carboxylic acid precursors malonyl-coenzyme A (CoA), (2S)-aminomalonyl-acyl carrier protein (ACP), and (2R)-hydroxymalonyl-ACP (4, 12). The proposal for l-Ser incorporation was made prior to the full elucidation of the stereochemistry of ZmA. The condensation of amino acids and carboxylic acids suggests that ZmA is assembled via a nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) megasynthase. These megasynthases are modular enzymes with a set of catalytic domains, or modules, for each precursor incorporated into the natural product (reviewed in references 13 and 40). Support for the involvement of this type of enzymology in ZmA biosynthesis comes from a combination of genetic and biochemical studies. Transposon mutagenesis of B. cereus UW85 identified insertions in genes coding for NRPS and PKS enzymology that abolished ZmA production (12). Sequencing of a 19-kb fragment of the ZmA biosynthesis gene cluster identified genes coding for NRPS and PKS enzymology (12). Furthermore, other groups investigating ZmA biosynthesis in B. thuringiensis strains have identified genes coding for NRPS modules that are essential for ZmA biosynthesis in these strains (35, 49, 50). Finally, we used biochemistry and mass spectrometry to establish the existence of two ACP-linked PKS extender units, (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP (4). All of these data support the hypothesis that the backbone of ZmA is assembled by an NRPS/PKS megasynthase.In addition to the mixed amino acid and carboxylic acid backbone, ZmA also contains a terminal amide (Fig. ​(Fig.1).1). How these amide groups are formed was investigated by Müller and colleagues and Silakowski and colleagues as they deciphered how myxothiazole is biosynthesized (29, 36). Briefly, the NRPS/PKS megasynthase that assembles the backbone of myxothiazole forms a product that is 1 amino acid longer than myxothiazole. This results in a biosynthetic intermediate that contains a glycyl residue at the C terminus of myxothiazole, while the intermediate remains thioesterified to the peptidyl carrier protein (PCP) domain of the terminal NRPS module. The α-carbon of the glycine is hydroxylated by a flavin-dependent monooxygenase, a modification that results in an unstable intermediate that spontaneously releases the myxothiazole backbone, with the nitrogen of the terminal amide coming from the glycine. The terminal PCP domain contains the glyoxyl group left after C-N bond cleavage, and this product is released from the PCP domain by the neighboring thioesterase (Te) domain. Based on this precedent, the terminal amide of ZmA may be produced by a similar mechanism.Here, we present the identification of the complete ZmA biosynthesis gene cluster from B. cereus UW85. The biosynthesis gene cluster was identified by locating the previously reported biosynthesis genes and by mapping the locations of transposon insertions that abolished the ability of B. cereus UW85 to produce ZmA. As expected, the gene cluster codes for NRPS and PKS enzymology that is likely to be involved in ZmA assembly from its amino acid and carboxylic acid precursors. Surprisingly, we fiound that ZmA not only is likely to be processed at its C terminus to generate the terminal amide by a mechanism similar to that seen in myxothiazole biosynthesis, but it appears to also be processed at its N terminus. These two processing events potentially lead to the biosynthesis of two additional metabolites besides ZmA. Furthermore, the kanosamine biosynthesis gene cluster appears to be fully contained within the ZmA biosynthesis gene cluster. A mechanism for ZmA production is presented, along with proposals for how three additional metabolites are produced by the enzymes encoded by this unusual gene cluster.     

The Gene Cluster for Spectinomycin Biosynthesis and the Aminoglycoside-Resistance Function of spcM in Streptomyces spectabilis

The gene cluster for spectinomycin biosynthesis from Streptomyces spectabilis was analyzed completely and registered under the accession number EU255259 at the National Center for Biotechnology Information. Based on sequence analysis, spcM of the S. spectabilis cluster is the only methyltransferase candidate required for methylation in spectinomycin biosynthesis. It has high similarity with the conserved domain of DNA methylase, which contains both N-4 cytosine-specific DNA methylases and N-6 adenine-specific DNA methylases. Nucleotide methylation can provide antibiotic resistance, such as 16S rRNA methyltransferase, to Enterobacteriaceae. We therefore tested a hypothesis that SpcM offers aminoglycoside resistance to bacteria. The heterologous expression of spcM in Escherichia coli and S. lividans enhanced resistance against spectinomycin and its relative aminoglycoside antibiotics. We therefore propose that one of the functions of SpcM may be conferring aminoglycoside antibiotic resistance to cells.     

Horizontal Transfer of the Photosynthesis Gene Cluster and Operon Rearrangement in Purple Bacteria

A 37-kb photosynthesis gene cluster was sequenced in a photosynthetic bacterium belonging to the beta subclass of purple bacteria (Proteobacteria), Rubrivivax gelatinosus. The cluster contained 12 bacteriochlorophyll biosynthesis genes (bch), 7 carotenoid biosynthesis genes (crt), structural genes for photosynthetic apparatuses (puf and puh), and some other related genes. The gene arrangement was markedly different from those of other purple photosynthetic bacteria, while two superoperonal structures, crtEF-bchCXYZ-puf and bchFNBHLM-lhaA-puhA, were conserved. Molecular phylogenetic analyses of these photosynthesis genes showed that the photosynthesis gene cluster of Rvi. gelatinosus was originated from those of the species belonging to the alpha subclass of purple bacteria. It was concluded that a horizontal transfer of the photosynthesis gene cluster from an ancestral species belonging to the alpha subclass to that of the beta subclass of purple bacteria had occurred and was followed by rearrangements of the operons in this cluster.     

Cloning of the Chlorothalonil-Degrading Gene Cluster and Evidence of Its Horizontal Transfer

Strain Ochrobactrum lupine TP-D1 was found to degrade chlorothalonil (TPN) to 4-hydroxy-chlorothalonil (TPN-OH). To clone the related degrading gene, genomic library of TP-D1 was constructed using Escherichia coli DH10B and two positive clones 889 and 838 were gained. However, no plasmid was detected in clone 889. And in clone 838, a 3494 bp fragment was cloned which contains a 984 bp hydrolytic dehalogenase (chd) gene and a 1926 bp insertion element IS-Olup. The insertion element contains a transposase coding region (1026 bp), an ATP-binding protein coding region (657 bp) and flanked by 20 bp inverted repeat sequences. Further isolation provided another seven TPN-degrading strains, they belonged to the genera of Pseudomonas sp., Achromobacter sp., Ochrobactrum sp., Ralstonia sp., and Lysobacter sp. PCR strategy showed that they all contain the same structure of chd gene and the upstream IS-Olup. Our evidences collectively suggest that chd gene may be disseminated through horizontal gene transfer based on phylogenetic analysis of the cluster and their host bacterial strains. At the same time, the chd gene was amplified from genome of the positive clone 889, which also provides some potential evidence to the gene horizontal transfer.     

Widespread Natural Occurrence of Hydroxyurea in Animals

Here we report the widespread natural occurrence of a known antibiotic and antineoplastic compound, hydroxyurea in animals from many taxonomic groups.Hydroxyurea occurs in all the organisms we have examined including invertebrates (molluscs and crustaceans), fishes from several major groups, amphibians and mammals. The species with highest concentrations was an elasmobranch (sharks, skates and rays), the little skate Leucoraja erinacea with levels up to 250 μM, high enough to have antiviral, antimicrobial and antineoplastic effects based on in vitro studies. Embryos of L. erinacea showed increasing levels of hydroxyurea with development, indicating the capacity for hydroxyurea synthesis. Certain tissues of other organisms (e.g. skin of the frog (64 μM), intestine of lobster (138 μM) gills of the surf clam (100 μM)) had levels high enough to have antiviral effects based on in vitro studies. Hydroxyurea is widely used clinically in the treatment of certain human cancers, sickle cell anemia, psoriasis, myeloproliferative diseases, and has been investigated as a potential treatment of HIV infection and its presence at high levels in tissues of elasmobranchs and other organisms suggests a novel mechanism for fighting disease that may explain the disease resistance of some groups. In light of the known production of nitric oxide from exogenously applied hydroxyurea, endogenous hydoxyurea may play a hitherto unknown role in nitric oxide dynamics.