Redox-Dependent Gene Regulation in Rhodobacter sphaeroides 2.4.1T: Effects on Dimethyl Sulfoxide Reductase (dor) Gene Expression

The ability of Rhodobacter sphaeroides 2.4.1^T to respire anaerobically with the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) is manifested by the molybdoenzyme DMSO reductase, which is encoded by genes of the dor locus. Previously, we have demonstrated that dor expression is regulated in response to lowered oxygen tensions and the presence of DMSO or TMAO in the growth medium. Several regulatory proteins have been identified as key players in this regulatory cascade: FnrL, DorS-DorR, and DorX-DorY. To further examine the role of redox potentiation in the regulation of dor expression, we measured DMSO reductase synthesis and β-galactosidase activity from dor::lacZ fusions in strains containing mutations in the redox-active proteins CcoP and RdxB, which have previously been implicated in the generation of a redox signal affecting photosynthesis gene expression. Unlike the wild-type strain, both mutants were able to synthesize DMSO reductase under strictly aerobic conditions, even in the absence of DMSO. When cells were grown photoheterotrophically, dorC::lacZ expression was stimulated by increasing light intensity in the CcoP mutant, whereas it is normally repressed in the wild-type strain under such conditions. Furthermore, the expression of genes encoding the DorS sensor kinase and DorR response regulator proteins was also affected by the ccoP mutation. By using CcoP-DorR and CcoP-DorY double mutants, it was shown that the DorR protein is strictly required for altered dor expression in CcoP mutants. These results further demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in R. sphaeroides and identify the DorS-DorR proteins as a redox-dependent regulatory system controlling dor expression.     

In Vivo Analysis of Cobinamide Salvaging in Rhodobacter sphaeroides Strain 2.4.1

The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B₁₂). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ⁺ strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.Cobamides, such as adenosylcobalamin (AdoCbl, coenzyme B₁₂), are a group of complex cobalt-containing cyclic tetrapyrrole cofactors whose biosynthesis by bacteria and archaea requires substantial genetic information (>25 genes) (reviewed in references 25, 47, and 56). Two pathways for the de novo synthesis of the corrin ring have been described on the basis of the timing of cobalt insertion into the ring. The late cobalt insertion or aerobic pathway has been well studied in Pseudomonas denitrificans (9), while the early cobalt insertion or anaerobic pathway has been best studied in Salmonella enterica serovar Typhimurium LT2 (25). Many organisms, including those that synthesize AdoCbl de novo, salvage incomplete corrinoids (e.g., cobinamide [Cbi]) from their environments and use them as precursors for the synthesis of complete cobamide cofactors. Cbi is not an intermediate of the de novo AdoCbl biosynthesis pathway but can be converted into one by a process known as Cbi salvaging (Fig. ​(Fig.1)1) (24).Open in a separate windowFIG. 1.Abbreviated view of cobinamide salvaging pathways. Corrin ring-containing intermediates are in bold text. The letter A indicates the de novo corrin ring biosynthesis pathway. Abbreviations: Ado-, adenosyl-; AP, 1-amino-2-propanol; AP-P, 1-amino-2-propanol-phosphate; CobB, hydrogenobyrinic acid a,c-diamide synthase; CobD, adenosylcobinamide-phosphate synthetase; CobP, NTP:adenosylcobinamide kinase, GTP:adenosylcobinamide-phosphate guanylyltransferase; CobY, GTP:adenosylcobinamide-phosphate guanylyltransferase; CbiZ, adenosylcobinamide amidohydrolase. Functional groups are indicated as follows: Me, methyl; Ac, acetamide; and Pr, propionamide.The first step of Cbi salvaging is adenosylation of the molecule to adenosylcobinamide (AdoCbi) (24). The adenosyltransferases which catalyze this reaction are broadly distributed throughout the three domains of life (13, 14, 20, 32, 38). Two distinct pathways for converting AdoCbi into an intermediate of the de novo AdoCbl biosynthesis pathway have been described for prokaryotes. One, which is to date found only in bacteria, relies on a bifunctional nucleoside triphosphate (NTP):AdoCbi kinase (EC 2.7.7.62), GTP:AdoCbi-phosphate (AdoCbi-P) guanylyltransferase (EC 2.7.1.156) enzyme (called CobP in P. denitrificans and CobU in S. Typhimurium), which phosphorylates AdoCbi to AdoCbi-P and converts AdoCbi-P to AdoCbi-GDP (10, 41, 55).Previous work from our laboratory has shown that archaea lack the bifunctional NTP:AdoCbi kinase, GTP:AdoCbi-P guanylyltransferase enzyme and rely on a second pathway for Cbi salvaging (54, 62). In this pathway, AdoCbi is converted to adenosylcobyric acid (AdoCby) by an AdoCbi amidohydrolase (EC 3.5.1.90) known as CbiZ (58, 59, 62). The conversion of AdoCbi-P to AdoCbi-GDP for de novo AdoCbl biosynthesis in archaea is catalyzed by a monofunctional GTP:AdoCbi-P guanylyltransferase (EC 2.7.7.62) called CobY (54, 60), which has not been found in any bacterium.We recently showed that a small percentage of bacterial genomes encode orthologs of both CobP-type and CbiZ-type Cbi salvaging enzymes, raising the question of why these organisms might contain two redundant Cbi salvaging systems (29). A phylogenetic analysis showed that CbiZ has its roots in the archaea and that the cbiZ gene was acquired by several bacterial lineages via horizontal gene transfer.We previously showed that the CbiZ and CobP enzymes from the photosynthetic alphaproteobacterium Rhodobacter sphaeroides are functional in vitro and in vivo in a heterologous complementation system (29). However, the question of how the two Cbi salvaging systems might function in R. sphaeroides remained unresolved.In this paper, we show that R. sphaeroides 2.4.1 synthesizes substantial amounts of cobalamin (Cbl) and that it salvages incomplete corrinoids from its environment. We present in vivo genetic evidence that both the bacterial-type CobP-dependent and archaeal-type CbiZ-dependent Cbi salvaging pathways are functional in this organism. This work represents the first in vivo genetic analysis of coenzyme B₁₂ synthesis and salvaging in R. sphaeroides.     

Photosynthesis genes and their expression in Rhodobacter sphaeroides 2.4.1: a tribute to my students and associates

This minireview traces the photosynthesis genes, their structure, function and expression in Rhodobacter sphaeroides 2.4.1, as applied to our understanding of the inducible photosynthetic intracytoplasmic membrane system or ICM. This focus has represented the research interests of this laboratory from the late 1960s to the present. This opportunity has been used to highlight the contributions of students and postdoctorals to this research effort. The work described here took place in a much greater and much broader context than what can be conveyed here. The ‘timeline’ begins with a clear acknowledgment of the work of June Lascelles and William Sistrom, whose foresight intuitively recognized the necessity of a ‘genetic’ approach to the study of photosynthesis in R. sphaeroides. The ‘timeline’ concludes with the completed genome sequence of R. sphaeroides 2.4.1. However, it is hoped the reader will recognize this event as not just a new beginning, but also as another hallmark describing this continuum. This revised version was published online in June 2006 with corrections to the Cover Date.     

Revised Sequence and Annotation of the Rhodobacter sphaeroides 2.4.1 Genome

The DNA sequences of chromosomes I and II of Rhodobacter sphaeroides strain 2.4.1 have been revised, and the annotation of the entire genomic sequence, including both chromosomes and the five plasmids, has been updated. Errors in the originally published sequence have been corrected, and ∼11% of the coding regions in the original sequence have been affected by the revised annotation.     

DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1

This paper describes the DNA sequence of the photosynthesis region of Rhodobacter sphaeroides 2.4.1^T. The photosynthesis gene cluster is located within a ~73 kb AseI genomic DNA fragment containing the puf, puhA, cycA and puc operons. A total of 65 open reading frames (ORFs) have been identified, of which 61 showed significant similarity to genes/proteins of other organisms while only four did not reveal any significant sequence similarity to any gene/protein sequences in the database. The data were compared with the corresponding genes/ORFs from a different strain of R.sphaeroides and Rhodobacter capsulatus, a close relative of R.sphaeroides. A detailed analysis of the gene organization in the photosynthesis region revealed a similar gene order in both species with some notable differences located to the pucBAC=cycA region. In addition, photosynthesis gene regulatory protein (PpsR, FNR, IHF) binding motifs in upstream sequences of a number of photosynthesis genes have been identified and shown to differ between these two species. The difference in gene organization relative to pucBAC and cycA suggests that this region originated independently of the photosynthesis gene cluster of R.sphaeroides.     

浑球红细菌（Rhodobacter sphaeroides）中氢化酶正调节基因hupR的克隆及功能分析

从光合细菌Rhodobacter sphaeroides基因文库中分离出含有氢化酶基因簇（hup）的粘粒cosmid 1后,亚克隆了R.sphaeroides的氢化酶调节基因hupR,测定了hupR的核苷酸序列,并完成了氢化酶基因簇的部分物理图谱。实验结果表明,hupR基因全长1476bp,编码的HupR基因分子量约为54.031kD(EMBL接受号：A243734)。与R.capsulatus中HupR相比,同源性高达73％。同源性比较结果表明,它属于双组分调节系统中受体蛋白。hupR基因在E.coli中进行了体外表达,纯化后测定得到的HupR蛋白 分子量大小与hupR基因推测的分子量大小一致。通过双交换,将卡那霉素抗性基因插入hupR基因,获得丧失氢化酶活性的hupR^-的突变株,KR5和KR7。hupS∷lacZ融合基因在野生型中的转录表达量是在该突变株中的7－9倍。将hupR基因置于弱启动子pfru下游,构建了质粒pNRC3,并将其导入hupR^-的突变株,可使突变株重新获得氢化酶活性。以上结果说明,HupR蛋白对氢化酶的转录表达起着正调节作用。在HupR蛋白的磷酸化区域进行定点和缺失突变。不影响HupR激活氢化酶基因的表达,推测HupR蛋白是在非磷酸化的状态下起调节作用的。     

类球红菌自诱导物合成酶基因cerI的克隆及其原核表达

根据类球红菌(Rhodobacter sphaeroides 2.4.1)自诱导物合成酶基因cerI的序列,设计并合成了1对特异性引物,在引物的5′和3′分别加入含有HindIII和XhoI限制性酶切位点的序列,以类球红细菌Rhodobacter sphaeroides基因组为模板扩增了cerI基因序列.将PCR产物与pMD18-T载体连接,转化大肠杆菌DH5α.鉴定成功获得目的片段,经HindIII和XhoI双酶切后与载体pET-28a(+)连接,构建原核表达质粒pET-28a(+)-cerI,并将其转化宿主菌BL21(DE3),用IPTG诱导其表达.SDS-PAGE分析表明,重组载体pET-28a(+)-cerI可成功地在大肠杆菌中表达cerI蛋白.     

光合细菌Rhodobacter sphaeroides新型表达系统研究进展

利用当前商业化的表达系统生产重组蛋白时,在寄主培养过程中都不能快速实时地检测重组蛋白的表达水平。光合细菌Rb.sphaeroides是研究细菌光合作用和膜蛋白形成的重要模式生物,具有开发成为一种新型表达系统的潜能。介绍了光合细菌Rb.sphaeroides新型表达系统的优势和开发情况。