Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed.     

The puhE gene of Rhodobacter capsulatus is needed for optimal transition from aerobic to photosynthetic growth and encodes a putative negative modulator of bacteriochlorophyll production

A conserved orf of previously unknown function (herein designated as puhE) is located 3' of the reaction centre H (puhA) gene in purple photosynthetic bacteria, in the order puhABCE in Rhodobacter capsulatus. Disruptions of R. capsulatus puhE resulted in a long lag in the growth of photosynthetic cultures inoculated with cells grown under high aeration, and increased the level of the peripheral antenna, light-harvesting complex 2 (LH2). The amount of the photosynthetic reaction centre (RC) and its core antenna, light-harvesting complex 1 (LH1), was reduced; however, there was no decrease in expression of a lacZ reporter fused to the puf (RC and LH1) promoter, in RC assembly in the absence of LH1, or in LH1 assembly in the absence of the RC. In strains that lack LH2, disruption of puhE increased the in vivo absorption at 780 nm, which we attribute to excess bacteriochlorophyll a (BChl) pigment production. This effect was seen in the presence and absence of PufQ, a protein that stimulates BChl biosynthesis. Expression of puhE from a plasmid reduced A(780) production in puhE mutants. We suggest that PuhE modulates BChl biosynthesis independently of PufQ, and that the presence of excess BChl in PuhE(-)LH2(+) strains results in excess LH2 assembly and also interferes with the adaptation of cells during the transition from aerobic respiratory to anaerobic photosynthetic growth.     

Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus.

By deletion of the pufX gene of Rhodobacter capsulatus from a plasmid carrying the puf operon and complementation of a chromosomal puf operon deletion, we created pufX mutants and used them to characterize possible functions of the pufX gene product. The pufX mutants were incapable of photosynthetic growth in a minimal medium, or in a rich medium at low light intensities, although second-site mutations suppressed this phenotype. Measurements made in vitro with intact and solubilized chromatophore preparations indicated that the individual complexes of the photosynthetic unit seemed to function normally, but electron transfer from the reaction center to the cytochrome b/c1 complex was impaired. The structures of the photosynthetic apparatus of pseudo-wild type and mutant strains were evaluated using absorption spectroscopy and electron microscopy. The pufX mutants had intracytoplasmic membrane invaginations about 50% larger in diameter than those of the pseudo-wild type and higher levels of B870 light-harvesting complex. It is concluded that the PufX protein plays an important role in the structure of the functional photosynthetic unit, and its absence results in loss of efficient electron transfer from the QB site of the reaction center to the Qz site of the cytochrome b/c1 complex.     

The single-stranded-DNA-binding protein encoded by the Escherichia coli F factor can complement a deletion of the chromosomal ssb gene.

Genes encoding single-stranded-DNA-binding proteins (SSBs) are carried by a variety of large self-transmissible plasmids, and it previously has been shown that these plasmid-borne genes can complement conditional lethal alleles of the ssb gene on the Escherichia coli chromosome for cellular viability. We have tested one of the plasmid-borne ssb genes, the ssf gene from the E. coli F factor, for its ability to complement total deletion of the chromosomal ssb gene for viability. We have found that ssf can complement the ssb deletion, but only when it is present on a high-copy-number plasmid. Cells that are totally dependent on the F-factor-encoded SSB for viability manifest growth properties indicative of problems in DNA replication.     

Characterization of a mutant strain of Rhodovulum sulfidophilum lacking the pufA and pufB genes encoding the polypeptides for the light-harvesting complex 1 (B 870)

Contradictory results on the effectiveness of energy transfer from the light harvesting complex 2 (LH2) directly to the reaction center (RC) in mutant strains lacking the core light-harvesting complex 1 (LH1) have been obtained with cells of Rhodobacter capsulatus and Rhodobacter sphaeroides. A LH1⁻ mutant of Rhodovulum sulfidophilum, named rsLRI, was constructed by deletion of the pufBA genes, resulting in a kanamycin resistant photosynthetically positive clone. To restore the wild type phenotype, a complemented strain C2 was constructed by inserting in trans a DNA segment containing the pufBA genes. Light-induced FTIR difference spectra indicate that the RC in the rsLRI mutant and in the C2 complemented strains are functionally and structurally identical with those in the wild type strain, demonstrating that the assembly and the function of the RC is not impaired by the LH1 deletion. The photosynthetic growth rate of the rsLRI strain increased with decreasing light intensity. At 50 W m⁻² no photosynthetic growth was observed. These results indicate that the light energy harvested by the LH2 complex was not or inefficiently transferred to the RC; thus most of the energy necessary for photosynthetic growth is in the LH1⁻ strain directly absorbed by the RC. It is supposed that in the mutant strain, RC and LH2 cannot interact in an efficient way.     

Assembly of the light-harvesting chlorophyll antenna in the green alga Chlamydomonas reinhardtii requires expression of the TLA2-CpFTSY gene

The truncated light-harvesting antenna2 (tla2) mutant of Chlamydomonas reinhardtii showed a lighter-green phenotype, had a lower chlorophyll (Chl) per-cell content, and higher Chl a/b ratio than corresponding wild-type strains. Physiological analyses revealed a higher intensity for the saturation of photosynthesis and greater P(max) values in the tla2 mutant than in the wild type. Biochemical analyses showed that the tla2 strain was deficient in the Chl a-b light-harvesting complex, and had a Chl antenna size of the photosystems that was only about 65% of that in the wild type. Molecular and genetic analyses showed a single plasmid insertion in the tla2 strain, causing a chromosomal DNA rearrangement and deletion/disruption of five nuclear genes. The TLA2 gene, causing the tla2 phenotype, was cloned by mapping the insertion site and upon complementation with each of the genes that were deleted. Successful complementation was achieved with the C. reinhardtii TLA2-CpFTSY gene, whose occurrence and function in green microalgae has not hitherto been investigated. Functional analysis showed that the nuclear-encoded and chloroplast-localized CrCpFTSY protein specifically operates in the assembly of the peripheral components of the Chl a-b light-harvesting antenna. In higher plants, a cpftsy null mutation inhibits assembly of both the light-harvesting complex and photosystem complexes, thus resulting in a seedling-lethal phenotype. The work shows that cpftsy deletion in green algae, but not in higher plants, can be employed to generate tla mutants. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.     

Effects of Precise Deletions in Rhodobacter sphaeroides Reaction Center Genes on Steady-state Levels of Reaction Center Proteins: A Revised Model for Reaction Center Assembly

Possible interactions between photosynthetic reaction center (RC) proteins that protect these membrane proteins from proteolytic digestion in RC complex assembly were evaluated by use of translationally in-frame (nonpolar) RC gene-specific deletions. The RC H, RC M and RC L proteins were produced from plasmids, either alone or in concert with one or both of the others, in a strain of Rhodobacter sphaeroides that contained chromosomal deletions of all three RC genes. The steady-state amounts of these proteins in cell membrane and soluble fractions were assessed in western blots. The data are used to propose a model of RC assembly in which the RC M protein accumulates in the cell membrane regardless of the presence of the RC H and RC L proteins, and the RC M protein is a nucleus for addition of RC L followed by RC H in assembly of the RC holocomplex.     

Construction of a gene bank of Rhodopseudomonas capsulata using a broad host range DNA cloning system

A gene bank of the phototrophic bacterium Rhodopseudomonas capsulata was constructed using the binary plasmid system pRK290/pRK2013. Fragments of about 20 kb of chromosomal DNA of R. capsulata strain 37b4 were inserted into the cloning vector pRK290. The hybrid plasmids of the gene bank, maintained in Escherichia coli HB101 were transferred by conjugation to R. capsulata strains defective in the photosynthetic apparatus with frequencies of 5×10^-4 to 5×10^-2. Phototrophically growing transconjugants occurred with frequencies of 5×10^-7 to 5×10^-6. Recombination between the hybrid plasmids and the R. capsulata chromosome was shown. The hybrid plasmid pRCF1002, carrying a 25 kb insert of R. capsulata wild type DNA, was isolated from one E. coli clone of the gene bank. It reconstituted some bacteriochlorophyll- and photosynthetic negative mutants to phototrophic growth.Abbreviations Bchl Bacteriochlorophyll - RC reaction center - LH light-harvesting complex - Crt carotenoid - pho phototrophic growth - P Bchl precursor excreted, the number behind P indicates the maximum of absorption in ether (nm) - SDS sodium dodecyl sulfate - Tc tetracycline - Km kanamycin - Gm gentamicin - r resistant - kb kilo base pairs Dedicated to Hans-Günter Schlegel on occasion of his 60th birthday     

Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis-acting function dispensable for chromosomal gene transfer

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra⁺ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.     

AmoA、AmoE和AmoF蛋白在嗜水气单胞菌铁载体合成中的作用研究

铁载体被认为是嗜水气单胞菌的毒力因子之一, 其由amoCEBFAGH七个基因编码, AmoCGH在前人的研究中已证实参与铁载体的合成。RT-PCR实验表明amoAEF基因的表达受到铁的调控。为进一步探究amoAEF基因的功能, 利用融合PCR和基因同源重组原理, 以自杀性质粒PRE112为载体构建基因缺失株ΔamoA、ΔamoE和ΔamoF。通过CAS平板检测实验以及arnow实验来检测野生株WT与各基因缺失突变株铁载体的合成情况, 并比较野生株与各缺失株在低铁培养基中的生长差异。结果显示, 成功构建了基因缺失株ΔamoA、ΔamoE和ΔamoF; 在富铁条件下, 基因缺失株ΔamoA、ΔamoE和ΔamoF的生长与野生株无显著性差异, 但在低铁条件下, 基因缺失株ΔamoA、ΔamoE和ΔamoF的生长能力、铁载体合成能力显著低于野生株。可见, amoA、amoE和amoF基因是嗜水气单胞菌铁载体合成的关键基因, 其缺失会导致细菌在低铁环境中的生长受到抑制。     

Camphor Plasmid-Mediated Chromosomal Transfer in Pseudomonas putida

Camphor-utilizing strains of Pseudomonas putida have been shown to carry the genetic information required for camphor degradation on a plasmid. The plasmid-carrying strains can serve as donors of both plasmid-borne and chromosomal genes. As recipients, plasmid-deleted strains are much superior to those carrying the camphor pathway genes. The transfer frequency of chromosomal, but not plasmid-borne, genes is markedly enhanced if the donor cells are irradiated with ultraviolet light followed by 3-h of growth on a rich medium in the dark. Recombinants selected for prototrophy are stable and most acquire the camphor (CAM) plasmid concomitantly; only a few of the Cam(+) recombinants inherit the donor's ability to transfer chromosomal genes at a high frequency. Transfer-defective mutations occur on the CAM plasmid, affecting both CAM and chromosomal gene transfer.     

Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene sodA.

We report the unexpected result that Escherichia coli isolates containing a multicopy plasmid (pDT1.5) carrying the manganese-superoxide dismutase gene sodA were more sensitive than the wild type to paraquat-mediated growth inhibition. The pDT1.5 locus responsible for the paraquat-sensitive phenotype was delimited to a 0.6-kilobase segment by transposon Tn5 mutagenesis. Moreover, superoxide dismutase activity was the same as in the wild type in strains carrying pDT1.5::Tn5 insertions mapping to the 0.6-kilobase locus. These data identify the 0.6-kilobase segment as the locus of sodA and establish an association between growth inhibition by paraquat and the function of the plasmid-borne sodA gene.     

Gene sequence encoding early enzymes of arginine synthesis within a cluster in Bacillus subtilis, as revealed by cloning in Escherichia coli.

From a partial Sau3A gene library of Bacillus subtilis chromosomal DNA in the expression plasmid pRK9, four hybrid plasmids were isolated carrying overlapping segments of the argA-argF-cpa cluster. The complementation patterns within Escherichia coli arginine auxotrophs of these hybrids and deletion derivatives provided the gene order argC-argA-argE-argB-argD-cpa-argF.     

rpoS基因在肠杆菌CGMCC 5087响应环境胁迫中的功能

[背景]苯乙醇(2-Phenylethanol,2-PE)是一种具有玫瑰香气味的高级香料添加剂,被广泛应用于香水、化妆品、食品和医药等领域.目前,利用工程菌合成苯乙醇有很好的应用前景.我们分离到一株肠杆菌(Enterobactersp.)CGMCC 5087,其可以通过苯丙酮酸途径合成2-PE.然而该菌的生长受到不同环...     

Method to integrate multiple plasmids into the mycobacterial chromosome

In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB. This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 integrase (int) gene. The integrated plasmid has a plasmid-borne attB site that is preserved and will accept the integration of additional mycobacterial plasmids containing the L5 attP site. This system should be useful in the construction of novel mycobacterial strains. In particular, this system provides a method by which several recombinant antigens or reporter constructs can be sequentially inserted into a mycobacterial strain and subsequently tested.