Gene transfer system for Rhodopseudomonas viridis.

A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2] thus far demonstrated in purple bacteria. It was also demonstrated that Rhizobium meliloti can be used as a donor for conjugation with R. viridis. From a genomic cosmid library of R. viridis constructed in the vector pHC79, clones that coded for subunits H (puh operon), L, M and cytochrome c (puf operon) of the photosynthetic reaction center were isolated and characterized. For linkage of the two operons on the genome, cosmids that overlapped with the operon-carrying clones were identified. The relative positions of the two operons could not be determined, but the operons must be more than 100 kilobase pairs apart. Thus, the genomic organization of the reaction center in R. viridis is different from that of Rhodobacter capsulatus, for which a distance of about 39 kilobase pairs was determined. From a spontaneous mutant of R. viridis that is resistant to the herbicide terbutryn, the puf operon was cloned in pKVS1 and transferred by conjugation into R. viridis wild-type cells. The resulting exconjugants were resistant to the herbicide, which demonstrated that the puf operon on pKVS1 constructions was functionally expressed in R. viridis.     

Genetic and structural organization of the aminophenol catabolic operon and its implication for evolutionary process

The aminophenol (AP) catabolic operon in Pseudomonas putida HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol meta cleavage of 2-aminophenol. The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene, nbzR, and the following nine structural genes, nbzJCaCbDGFEIH, which encode catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain. It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form. A comparative study of the AP catabolic operon with other meta cleavage operons led us to suggest that the regulatory unit (nbzR) was derived from the MarR family and that the structural unit (nbzJCaCbDGFEIH) has evolved from the ancestral meta cleavage gene cluster. It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer.     

Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in the Delaware River

Photosynthesis genes and operons of aerobic anoxygenic photosynthetic (AAP) bacteria have been examined in a variety of marine habitats, but genomic information about freshwater AAP bacteria is lacking. The goal of this study was to examine photosynthesis genes of AAP bacteria in the Delaware River. In a fosmid library, we found two clones bearing photosynthesis gene clusters with unique gene content and organization. Both clones contained 37 open reading frames, with most of those genes encoding known AAP bacterial proteins. The genes in one fosmid were most closely related to those of AAP bacteria in the Rhodobacter genus. The genes of the other clone were related to those of freshwater beta-proteobacteria. Both clones contained the acsF gene, which is required for aerobic bacteriochlorophyll synthesis, suggesting that these bacteria are not anaerobes. The beta-proteobacterial fosmid has the puf operon B-A-L-M-C and is the first example of an uncultured bacterium with this operon structure. The alpha-3-proteobacterial fosmid has a rare gene order (Q-B-A-L-M-X), previously observed only in the Rhodobacter genus. Phylogenetic analyses of photosynthesis genes revealed a possible freshwater cluster of AAP beta-proteobacteria. The data from both Delaware River clones suggest there are groups of freshwater or estuarine AAP bacteria distinct from those found in marine environments.     

Computational analysis of Ciona intestinalis operons

Operons are clusters of genes that are co-regulated from a common promoter. Operons are typically associated with prokaryotes, although a small number of eukaryotes have been shown to possess them. Among metazoans, operons have been extensively characterized in the nematode Caenorhabditis elegans in which ～15% of the total genes are organized into operons. The most recent genome assembly for the ascidian Ciona intestinalis placed ～20% of the genes (2909 total) into 1310 operons. The majority of these operons are composed of two genes, while the largest are composed of six. Here is reported a computational analysis of the genes that comprise the Ciona operons. Gene ontology (GO) terms were identified for about two-thirds of the operon-encoded genes. Using the extensive collection of public EST libraries, estimates of temporal patterns of gene expression were generated for the operon-encoded genes. Lastly, conservation of operons was analyzed by determining how many operon-encoded genes were present in the ascidian Ciona savignyi and whether these genes were organized in orthologous operons. Over 68% of the operon-encoded genes could be assigned one or more GO terms and 697 of the 1310 operons contained genes in which all genes had at least one GO term. Of these 697 operons, GO terms were shared by all of the genes within 146 individual operons, suggesting that most operons encode genes with unrelated functions. An analysis of operon gene expression from nine different EST libraries indicated that for 587 operons, all of the genes that comprise an individual operon were expressed together in at least one EST library, suggesting that these genes may be co-regulated. About 50% (74/146) of the operons with shared GO terms also showed evidence of gene co-regulation. Comparisons with the C. savignyi genome identified orthologs for 1907 of 2909 operon genes. About 38% (504/1310) of the operons are conserved between the two Ciona species. These results suggest that like C. elegans, operons in Ciona are comprised of a variety of genes that are not necessarily related in function. The genes in only 50% of the operons appear to be co-regulated, suggesting that more complex gene regulatory mechanisms are likely operating.     

Bacterial regulatory networks include direct contact of response regulator proteins: interaction of RegA and NtrX in Rhodobacter capsulatus

The formation of photosynthetic complexes in facultatively photosynthetic bacteria is controlled by the oxygen tension in the environment. In Rhodobacter capsulatus the two-component system RegB/RegA plays a major role in the redox control of photosynthesis genes but also controls other redox-dependent systems. The response regulator RegA is phosphorylated under low oxygen tension and activates the puf and puc operons, which encode pigment binding proteins, by binding to their promoter regions. Data from a yeast two-hybrid analysis as well as an in vitroanalysis indicate that RegA interacts with the NtrX protein, the response regulator of the NtrY/NtrX two-component system which is believed to be involved in regulation of nitrogen fixation genes. Our further analysis revealed that NtrX is indeed involved in the regulation of the puf and puc operons. Furthermore, we showed that an altered NtrX protein, which is predicted to adopt the conformation of phosphorylated NtrX protein, binds within the puf promoter region close to the RegA binding sites. We conclude that a direct interaction of two response regulators connects the regulatory systems for redox control and nitrogen control.     

Evidence against the selfish operon theory

According to the selfish operon hypothesis, the clustering of genes and their subsequent organization into operons is beneficial for the constituent genes because it enables the horizontal gene transfer of weakly selected, functionally coupled genes. The majority of these are expected to be non-essential genes. From our analysis of the Escherichia coli genome, we conclude that the selfish operon hypothesis is unlikely to provide a general explanation for clustering nor can it account for the gene composition of operons. Contrary to expectations, essential genes with related functions have an especially strong tendency to cluster, even if they are not in operons. Moreover, essential genes are particularly abundant in operons.     

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.