Isolation and characterization of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides.

The rpoN gene from Rhodobacter sphaeroides was isolated from a genomic library via complementation of a Rhodobacter capsulatus rpoN mutant. The rpoN gene was located on a 7.5-kb HindIII-EcoRI fragment. A Tn5 insertion analysis of this DNA fragment showed that a minimal DNA fragment of 5.3 kb was required for complementation. Nucleotide sequencing of the complementing region revealed the presence of nifUSVW genes upstream from rpoN. The rpoN gene was mutagenized via insertion of a gene encoding kanamycin resistance. The resulting rpoN mutant was not impaired in diazotrophic growth and was in all respects indistinguishable from the wild-type strain. Southern hybridizations using the cloned rpoN gene as a probe indicated the presence of a second rpoN gene. Deletion of the nifUS genes resulted in strongly reduced diazotrophic growth. Two conserved regions were identified in a NifV LeuA amino acid sequence alignment. Similar regions were found in pyruvate carboxylase and oxaloacetate decarboxylase. It is proposed that these conserved regions represent keto acid-binding sites.     

"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.     

Fine tuning bacterial chemotaxis: analysis of Rhodobacter sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and cheY genes

Rhodobacter sphaeroides chemotaxis is significantly more complex than that of enteric bacteria. Rhodobacter sphaeroides has multiple copies of chemotaxis genes (two cheA, one cheB, two cheR, three cheW, five cheY but no cheZ), controlling a single 'stop-start' flagellum. The growth environment controls the level of expression of different groups of genes. Tethered cell analysis of mutants suggests that CheY(4) and CheY(5) are the motor-binding response regulators. The histidine protein kinase CheA(2) mediates an attractant ('normal') response via CheY(4), while CheA(1) and CheY(5) appear to mediate a repellent ('inverted') response. CheY(3) facilitates signal termination, possibly acting as a phosphate sink, although CheY(1) and CheY(2) can substitute. The normal and inverted responses may be initiated by separate sets of chemoreceptors with their relative strength dependent on growth conditions. Rhodobacter sphaeroides may use antagonistic responses through two chemosensory pathways, expressed at different levels in different environments, to maintain their position in a currently optimum environment. Complex chemotaxis systems are increasingly being identified and the strategy adopted by R.sphaeroides may be common in the bacterial kingdom.     

Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification

Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene.     

Relationship between multiple copies of a T. brucei variable surface glycoprotein gene whose expression is not controlled by duplication

Unlike many other T. brucei variable surface glycoprotein (VSG) genes, the IITat 1.3 gene is not duplicated when it is expressed. Analysis of the multiple copies of this gene present in all IITaR 1 trypanosome clones by restriction enzyme mapping and sequencing shows that the expressed copy may have arisen by duplication and transposition to a telomeric site, as is observed for those VSG genes whose expression is linked to duplication. The existence of a mechanism selecting between a number of complete telomeric VSG gene copies for expression is implied by these results. Comparisons of the nontelomeric copies of the IITat 1.3 gene are consistent with involvement of gene duplication and mutational drift in the evolution of new VSG genes.     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.     

Rapid evolution through gene duplication and subfunctionalization of the testes-specific alpha4 proteasome subunits in Drosophila

Gene duplication is an important mechanism for acquiring new genes and creating genetic novelty in organisms. Evidence suggests that duplicated genes are retained at a much higher rate than originally thought and that functional divergence of gene copies is a major factor promoting their retention in the genome. We find that two Drosophila testes-specific alpha4 proteasome subunit genes (alpha4-t1 and alpha4-t2) have a higher polymorphism within species and are significantly more diverged between species than the somatic alpha4 gene. Our data suggest that following gene duplication, the alpha4-t1 gene experienced relaxed selective constraints, whereas the alpha4-t2 gene experienced positive selection acting on several codons. We report significant heterogeneity in evolutionary rates among all three paralogs at homologous codons, indicating that functional divergence has coincided with genic divergence. Reproductive subfunctionalization may allow for a more rapid evolution of reproductive traits and a greater specialization of testes function. Our data add to the increasing evidence that duplicated genes experience lower selective constraints and in some cases positive selection following duplication. Newly duplicated genes that are freer from selective constraints may provide a mechanism for developing new interactions and a pathway for the evolution of new genes.     

Biochemical study of multiple CheY response regulators of the chemotactic pathway of Rhodobacter sphaeroides

The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac(32)P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.     

In silico sequence analysis of arylamine N-acetyltransferases: evidence for an absence of lateral gene transfer from bacteria to vertebrates and first description of paralogs in bacteria

The arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes responsible for the biotransformation of various arylamine and heterocyclic amines, including drugs and carcinogenic compounds. NAT and NAT-like genes have been identified in several vertebrate and eubacterial species. Little is known about their evolutionary history, but the horizontal transfer of NAT genes from bacteria to vertebrates was recently suggested [S. Salzberg, O. White, J. Peterson, J. Eisen, Science 292 (2001) 1903]. We used various bioinformatics-based approaches to screen eukaryotic and prokaryotic genomes. We identified Mesorhizobium loti NAT genes as the first examples of NAT paralogs in prokaryotes. As shown for vertebrate species, the existence of NAT paralogs in this bacterium may be accounted for by enzymatic specialization after gene duplication. Phylogenetic analysis following the identification of a NAT ortholog in the nonvertebrate species Ciona intestinalis indicated that NAT genes are unlikely to be examples of direct horizontal gene transfer (HGT). Our study suggests that NAT genes have evolved from a common ancestor, with a succession of nonvertebrate intermediates. The absence of NAT genes in yeast, nematode worms, fruit flies, and mustard weed may result from gene loss in these nonvertebrate lineages. These results provide new insight into the taxonomic distribution and evolutionary history of this class of drug-metabolizing enzymes.     

Horizontal gene transfer of a plastid gene in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae)

Plastid sequences are among the most widely used in phylogenetic and phylogeographic studies in flowering plants, where they are usually assumed to evolve like non-recombining, uniparentally transmitted, single-copy genes. Among others, this assumption can be violated by intracellular gene transfer (IGT) within cells or by the exchange of genes across mating barriers (horizontal gene transfer, HGT). We report on HGT of a plastid region including rps2, trnL-F, and rbcL in a group of non-photosynthetic flowering plants. Species of the parasitic broomrape genus Phelipanche harbor two copies of rps2, a plastid ribosomal gene, one corresponding to the phylogenetic position of the respective species, the other being horizontally acquired from the related broomrape genus Orobanche. While the vertically transmitted copies probably reside within the plastid genome, the localization of the horizontally acquired copies is not known. With both donor and recipient being parasitic plants, a possible pathway for the exchange of genetic material is via a commonly attacked host.     

Expression of the nif and ntr genes of Klebsiella pneumoniae in Rhodobacter sphaeroides cells

The genes glnA, ntr, nif or their promoters from Klebsiella pneumoniae cloned on the vectors, based on the plasmid RSF1010, were introduced into Rhodobacter sphaeroides cells. It was found that K. pneumoniae genes glnA, nifB, nifE, nifL and nifH are not expressed in R. sphaeroides. Neither was the glnA gene from cyanobacterium Anabaena 7120 expressed in R. sphaeroides. No functional activity of K. pneumoniae product of ntrA gene which is expressed from its own promoter, and the product of the gene nifA which is expressed from the constitutive promoter of the kanamycin resistance gene of the transposon Tn903, was detected. The implications of these findings are discussed.     

Cloning and expression of the d-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli

Abstract Both form I and II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were detected in Thiobacillus intermedius by heterologous hybridization using specific probes from Anacystis nidulans and Rhodobacter sphaeroides , respectively. However, only the previously reported from I enzyme could be demonstrated in cells grown under a number of different conditions. The reason(s) why the form II gene is not expressed in T. intermedius is/are not clear at this time. The form II gene was isolated from a lambda library by screening with the Rb. sphaeroides probe. A Sal I fragment from this clone was ligated into pUC8 and transformed into Escherichia coli DH5α. Subclones pTi20IIA and pTi20IIB representing both orientations relative to the lac promoter were isolated. Low levels of RuBisCO activity were detected in both induced and non-induced pTi20IIA indicating the probable expression from a T. intermedius promoter. Induced pTi20IIB produced much higher levels of enzyme activity. Analysis of cell-free extracts using sucrose density gradients confirmed the expression of a form II RuBisCO similar in size to that found in Rhodobacter capsulatus . Other Calvin cycle genes were not clustered with either the form I or form II genes.     

Horizontally acquired cysteine synthase genes undergo functional divergence in lepidopteran herbivores

Horizontal gene transfer (HGT) plays an important role in evolutionary processes as organisms adapt to their environments, and now cases of gene duplication after HGT in eukaryotes are emerging at an increasing rate. However, the fate and roles of the duplicated genes over time in eukaryotes remain unclear. Here we conducted a comprehensive analysis of the evolution of cysteine synthase (CYS) in lepidopteran insects. Our results indicate that HGT-derived CYS genes are widespread and have undergone duplication following horizontal transfer in many lepidopteran insects. Moreover, lepidopteran CYS proteins not only have β-cyanoalanine synthase activity but also possess cysteine synthase activity that is involved in sulfur amino acid biosynthesis. Duplicated CYS genes show marked divergence in gene expression patterns and enzymatic properties, suggesting that they probably have undergone subfunctionalization and/or neofunctionalization in Lepidoptera. The gene transfer of CYS genes and subsequent duplication appears to have facilitated the adaptation of lepidopteran insects to different diets and promoted their ecological diversification. Overall, this study provides valuable insights into the ecological and evolutionary contributions of CYS in lepidopteran insects.Subject terms: Molecular ecology, Molecular evolution     

Evolutionary constraints and expression analysis of gene duplications in Rhodobacter Sphaeroides 2.4.1

ABSTRACT: BACKGROUND: Gene duplication is a major force that contributes to the evolution of new metabolic functions in all organisms. Rhodobacter sphaeroides 2.4.1 is a bacterium that displays a wide degree of metabolic versatility and genome complexity and therefore is a fitting model for the study of gene duplications in bacteria. A comprehensive analysis of 234 duplicate gene-pairs in R. sphaeroides was performed using structural constraint and expression analysis. RESULTS: The results revealed that most gene-pairs in in-paralogs are maintained under negative selection (omega [less than or equal to] 0.3), but the strength of selection differed among in-paralog gene-pairs. Although in-paralogs located on different replicons are maintained under purifying selection, the duplicated genes distributed between the primary chromosome (CI) and the second chromosome (CII) are relatively less selectively constrained than the gene-pairs located within each chromosome. The mRNA expression patterns of duplicate gene-pairs were examined through microarray analysis of this organism grown under seven different growth conditions. Results revealed that that ~62% of paralogs have similar expression patterns (cosine [greater than or equal to] 0.90) over all of these growth conditions, while only ~7% of paralogs are very different in their expression patterns (cosine < 0.50). CONCLUSIONS: The overall findings of the study suggest that only a small proportion of paralogs contribute to the metabolic diversity and the evolution of novel metabolic functions in R. sphaeroides. In addition, the lack of relationships between structural constraints and gene-pair expression suggests that patterns of gene-pair expression are likely associated with conservation or divergence of gene-pair promoter regions and other coregulation mechanisms.     

Analysis of the role of the nnrR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide.

Rhodobacter sphaeroides 2.4.1, which is incapable of denitrification, has been found to carry nnrR, the nor operon, and nnrS, which are utilized for denitrification in R. sphaeroides 2.4.3. The gene encoding nitrite reductase was not found in 2.4.1. Expression of beta-galactosidase activity from a norB-lacZ fusion was activated when cells of 2.4.1 were incubated with NO-producing bacteria. This result indicates that the products of nnrR and the genes flanking it are utilized when 2.4.1 is growing in an environment where denitrification occurs.     

DNA sequence duplication in Rhodobacter sphaeroides 2.4.1: evidence of an ancient partnership between chromosomes I and II

The complex genome of Rhodobacter sphaeroides 2.4.1, composed of chromosomes I (CI) and II (CII), has been sequenced and assembled. We present data demonstrating that the R. sphaeroides genome possesses an extensive amount of exact DNA sequence duplication, 111 kb or approximately 2.7% of the total chromosomal DNA. The chromosomal DNA sequence duplications were aligned to each other by using MUMmer. Frequency and size distribution analyses of the exact DNA duplications revealed that the interchromosomal duplications occurred prior to the intrachromosomal duplications. Most of the DNA sequence duplications in the R. sphaeroides genome occurred early in species history, whereas more recent sequence duplications are rarely found. To uncover the history of gene duplications in the R. sphaeroides genome, 44 gene duplications were sampled and then analyzed for DNA sequence similarity against orthologous DNA sequences. Phylogenetic analysis revealed that approximately 80% of the total gene duplications examined displayed type A phylogenetic relationships; i.e., one copy of each member of a duplicate pair was more similar to its orthologue, found in a species closely related to R. sphaeroides, than to its duplicate, counterpart allele. The data reported here demonstrate that a massive level of gene duplications occurred prior to the origin of the R. sphaeroides 2.4.1 lineage. These findings lead to the conclusion that there is an ancient partnership between CI and CII of R. sphaeroides 2.4.1.