The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.     

Importance of widespread gene transfer agent genes in alpha-proteobacteria

The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) is a model for several virus-like elements that seem to function solely for mediating gene exchange. Several genes that encode RcGTA are clearly related to bacteriophage genes but the cellular regulatory mechanisms that control RcGTA production indicate that RcGTA is more than just a defective prophage. Genome sequencing projects show that seemingly functional RcGTA-like structural gene clusters are present in many other species of alpha-proteobacteria, which might also produce RcGTA-like particles. Here, we use the genomic sequence data that are currently available to identify candidate GTA-producing species and propose an evolutionary scheme for RcGTA-like elements in the alpha-proteobacteria.     

Conservation of the photosynthesis gene cluster in Rhodospirillum centenum

Intraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photosynthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction-centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.     

"Green-like" and "red-like" RubisCO cbbL genes in Rhodobacter azotoformans

We found that Rhodobacter azotoformans IFO 16436T contains two different cbbL genes coding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunits. One gene is located within a "green-like" group of the RubisCO phylogenetic tree, and the other is located within a "red-like" group. This is the first report that one organism contains both green-like and red-like RubisCO genes. Moreover, by PCR using primers which amplify two green-like and red-like cbbL genes alternatively and dot blot hybridization, we demonstrated that Rhodobacter blasticus, Rhodobacter capsulatus, and Rhodobacter veldkampii possess only green-like cbbL genes, and Rhodobacter sphaeroides possesses only a red-like cbbL gene. In the cbbL phylogenic analysis, R. spaeroides and R. azotoformans 1 (red-like) formed a cluster within the red-like group, and R. capsulatus, R. azotoformans 2 (green-like), R. blasticus, and R. veldkampii formed a cluster within the green-like group. This suggests that red-like cbbL genes of Rhodobacter species were derived from one ancestor, and green-like cbbL genes were derived from another ancestor. On the other hand, molecular phylogeny of the bacteria indicates that R. veldkampii, which has only a green-like cbbL gene, is the earliest evolved Rhodobacter species and that R. azotoformans and R. sphaeroides, which have red-like cbbL genes, are the latest evolved. Consequently, the following hypothesis is proposed: the common ancestor of Rhodobacter had a green-like cbbL gene, the common ancestor of R. azotoformans and R. sphaeroides subsequently obtained a red-like cbbL gene by a horizontal gene transfer, and the ancestor of R. sphaeroides later lost the green-like cbbL gene.     

Environmental Factors Influencing Gene Transfer Agent (GTA) Mediated Transduction in the Subtropical Ocean

Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the marine environment with the strains examined is favored during times of elevated bacterial and GTA abundance as well as in areas of higher salinity.     

Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of Escherichia coli.

The fruK gene encoding fructose-1-phosphate kinase (FruK), located within the fructose (fru)-catabolic operon of Rhodobacter capsulatus, was sequenced. FruK of R. capsulatus (316 amino acids; molecular weight = 31,232) is the same size as and is homologous to FruK of Escherichia coli, phosphofructokinase B (PfkB) of E. coli, phosphotagatokinase of Staphylococcus aureus, and ribokinase of E. coli. These proteins therefore make up a family of homologous proteins, termed the PfkB family. A phylogenetic tree for this new family was constructed. Sequence comparisons plus chemical inactivation studies suggested the lack of involvement of specific residues in catalysis. Although the Rhodobacter FruK differed markedly from the other enzymes within the PfkB family with respect to amino acid composition, these enzymes exhibited similar predicted secondary structural features. A large internal segment of the Rhodobacter FruK was found to be similar in sequence to the domain bearing the sugar bisphosphate-binding region of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase of plants and bacteria. Proteins of the PfkB family did not exhibit statistically significant sequence identity with PfkA of E. coli. PfkA, however, is homologous to other prokaryotic and eukaryotic ATP- and PPi-dependent Pfks (the PfkA family). These eukaryotic, ATP-dependent enzymes each consist of a homotetramer (mammalian) or a heterooctamer (yeasts), with each subunit containing an internal duplication of the size of the entire PfkA protein of E. coli. In some of these enzymes, additional domains are present. A phylogenetic tree was constructed for the PfkA family and revealed that the bacterial enzymes closely resemble the N-terminal domains of the eukaryotic enzyme subunits whereas the C-terminal domains have diverged more extensively. The PPi-dependent Pfk of potato is only distantly related to the ATP-dependent enzymes. On the basis of their similar functions, sizes, predicted secondary structures, and sequences, we suggest that the PfkA and PfkB families share a common evolutionary origin.     

Phylogenetic analysis of the triterpene cyclase protein family in prokaryotes and eukaryotes suggests bidirectional lateral gene transfer

Functional constraints to modifications in triterpene cyclase amino acid sequences make them good candidates for evolutionary studies on the phylogenetic relatedness of these enzymes in prokaryotes as well as in eukaryotes. In this study, we used a set of identified triterpene cyclases, a group of mainly bacterial squalene cyclases and a group of predominantly eukaryotic oxidosqualene cyclases, as seed sequences to identify 5288 putative triterpene cyclase homologues in publicly available databases. The Cluster Analysis of Sequences software was used to detect groups of sequences with increased pairwise sequence similarity. The sequences fall into two main clusters, a bacterial and a eukaryotic. The conserved, informative regions of a multiple sequence alignment of the family were used to construct a neighbour-joining phylogenetic tree using the AsaturA and maximum likelihood phylogenetic tree using the PhyML software. Both analyses showed that most of the triterpene cyclase sequences were similarly grouped to the accepted taxonomic relationships of the organism the sequences originated from, supporting the idea of vertical transfer of cyclase genes from parent to offspring as the main evolutionary driving force in this protein family. However, a small group of sequences from three bacterial species ( Stigmatella , Gemmata and Methylococcus ) grouped with an otherwise purely eukaryotic cluster of oxidosqualene cyclases, while a small group of sequences from seven fungal species and a sequence from the fern Adiantum grouped consistently with a cluster of otherwise purely bacterial squalene cyclases. This suggests that lateral gene transfer may have taken place, entailing a transfer of oxidosqualene cyclases from eukaryotes to bacteria and a transfer of squalene cyclase from bacteria to an ancestor of the group of Pezizomycotina fungi.     

Structural and Functional Analyses of Photosynthetic Regulatory Genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus

Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans, species that demonstrate weak or no oxygen repression of photosystem synthesis. This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from Rhodobacter capsulatus and Rhodobacter sphaeroides. These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system. The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif. The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen. A Rhodovulum sulfidophilum mutant lacking regA or regB was constructed. These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in Rhodobacter species. Rhodobacter capsulatus regA- or regB-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with reg genes from Rhodovulum sulfidophilum and Roseobacter denitrificans. These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.     

Nucleotide sequence of the methoxyneurosporene dehydrogenase gene from Rhodobacter sphaeroides: comparison with other bacterial carotenoid dehydrogenases.

The nucleotide sequence of the 1794-bp fragment containing the crtD gene from Rhodobacter sphaeroides 2.4.1 encoding for methoxyneurosporene dehydrogenase has been determined. A 63% sequence identity was found when compared with the nucleotide sequence of the crtD gene from Rhodobacter capsulatus. A putative regulatory palindromic motif present in the crtD gene from R. capsulatus also exists in this gene from R. sphaeroides. The translated open reading frame of the crtD gene of R. sphaeroides has identified a polypeptide of 495 amino acids which shares a 56% sequence identity with the same CrtD protein of R. capsulatus. The N- and C-termini of these CrtD proteins present a high degree of similarity with the N- and C-termini of other carotenoid dehydrogenases including those encoded by crtI genes. This is in good agreement with the previously hypothesized homology between CrtI and CrtD proteins.     

Complete DNA sequence, specific Tn5 insertion map, and gene assignment of the carotenoid biosynthesis pathway of Rhodobacter sphaeroides.

The carotenoid biosynthesis genes form a cluster within the genome of Rhodobacter sphaeroides, lying in the middle of a larger cluster and 45 kb in length, which contains genes for bacteriochlorophyll biosynthesis and for the reaction center and light-harvesting apoproteins. The positions and approximate limits of the carotenoid genes were determined previously by localized transposon Tn5 mutagenesis and by comparison with the closely related Rhodobacter capsulatus carotenoid gene cluster. In this report, analysis of the DNA and deduced amino acid sequences of the carotenoid genes in R. sphaeroides are presented. Twenty-five Tn5 insertion mutants were used to produce a base-specific Tn5 insertion map of this region, and carotenoid gene assignment was supported by spectroscopic, ultrastructural, and high-pressure liquid chromatography analyses of these mutants. A region in the 3' end of crtD which affects bacteriochlorophyll biosynthesis was discovered, and CrtA was found to possess a proline-rich C-terminal region containing a repeated (Ala-Pro)n motif. CrtF also showed a high degree of sequence conservation with eukaryotic O-methyltransferases. This study provides gene sequences and assignments based upon a comprehensive structural, spectroscopic, and biochemical analysis of a range of carotenoid biosynthetic mutants; in each mutation, the point of Tn5 insertion is determined accurate to 1 bp on the gene cluster.     

草菇GH61家族基因的生物信息学分析及金属离子对其表达水平的影响

在前期工作中发现草菇含有30个GH61家族基因同源物（Vv_gh61_1至Vv_gh61_30）,进一步分析了这些基因的结构特点以及其编码蛋白基本性质和系统进化关系,并研究了Cu2+和Mn2+对基因表达水平的影响。分析表明有17个草菇GH61基因串联成6个基因簇,存在明显的串联重复现象,系统发育树与基因外显子位置分析表明串联重复基因分布在同一进化分枝上并具有相似的基因结构,串联重复基因编码序列一致性在71%–94%之间。草菇GH61编码蛋白的氨基酸数目在217–442aa之间,分子量和等电点分别介于22.4–45.4kDa和5.2–9.3之间,绝大多数都含有信号肽并定位在细胞外,都含有Glyco_hydro_61功能域以及CBM1、peroxidase等多样化的功能域,系统进化树表明草菇GH61家族基因具有3个主要进化分枝,与灰盖鬼伞等的GH61基因有较近的进化关系。金属离子诱导作用显示,Mn2+对草菇GH61家族基因的表达水平存在诱导作用而Cu2+的诱导作用不明显。     

Evolutionary genetics of the proline permease gene (putP) and the control region of the proline utilization operon in populations of Salmonella and Escherichia coli.

Virtually complete sequences (1,467 bp) of the proline permease gene (putP) and complete sequences (416 to 422 bp) of the control region of the proline utilization operon were determined for 16 strains of Salmonella, representing all eight subspecies, and 13 strains of Escherichia coli recovered from natural populations. Strains of Salmonella and E. coli differed, on average, at 16.3% of putP nucleotide sites and 17.5% of control region sites; the average difference between strains was much larger for Salmonella strains (4.6% of putP sites and 3.4% of control region sites) than for E. coli (2.4 and 0.9%, respectively). There was no difference in the distribution of polymorphic amino acid positions between the membrane-spanning and loop regions of the permease molecule, and rates of synonymous nucleotide substitution were virtually the same for the two domains. Statistical analysis yielded evidence of three probable cases of intragenic recombination, including the acquisition of a large segment of putP by strains of Salmonella subspecies VII from an unidentified source, the exchange of a 21-bp segment between two strains of E. coli, and the acquisition by one strain of E. coli of a cluster of 14 unique polymorphic control region sites from an unknown donor. An evolutionary tree for the putP and control region sequences was generally concordant with a tree for the gapA gene and a tree based on multilocus enzyme electrophoresis, thus providing evidence that for neither gene nor for enzyme genes in general has recombination occurred at rates sufficiently high or over regions sufficiently large to completely obscure phylogenetic relationships dependent on mutational divergence. It is suggested that the recombination rate varies among genes in relation to functional type, being highest for genes encoding cell surface and other proteins for which there is an adaptive advantage in structural diversity.     

孔石莼(Ulva pertusa)质体蓝素基因的克隆及基因特征分析(英文)

采用cDNA末端快速扩增的办法,从孔石莼(Ulva pertusa)中克隆获得质体蓝素基因。该基因完整的cDNA为787bp,包括40 bp 5’端非编码区和327 bp的3’端非编码区,以及一个420 bp的开放阅读框架,编码139个氨基酸的蛋白质。该基因编码质体蓝素的前体肽,其N端41个氨基酸残基为信号肽,后面为98个氨基酸残基的成熟肽。从Genbank中选择了13个质体蓝素的前体肽基因进行序列比对分析和构建进化树。孔石莼质体蓝素基因与其它质体蓝素基因的同源性为48.2%至78.8%。该进化树将来源于6种藻类植物的7个质体蓝素基因聚类在一起,显示出它们较近的进化关系。同样,也表现出11种生物的分子进化关系。序列比对结果显示,在质体蓝素的基因序列中存在两个高度保守的基序,它编码质体蓝素蛋白的铜结合活性位点。