Eustigmatophytes, a class of stramenopile algae (ochrophytes), include not only the extensively studied biotechnologically important genus Nannochloropsis but also a rapidly expanding diversity of lineages with much less well characterized biology. Recent discoveries have led to exciting additions to our knowledge about eustigmatophytes. Some proved to harbor bacterial endosymbionts representing a novel genus, Candidatus Phycorickettsia, and an operon of unclear function (ebo) obtained by horizontal gene transfer from the endosymbiont lineage was found in the plastid genomes of still other eustigmatophytes. To shed more light on the latter event, as well as to generally improve our understanding of the eustigmatophyte evolutionary history, we sequenced plastid genomes of seven phylogenetically diverse representatives (including new isolates representing undescribed taxa). A phylogenomic analysis of plastid genome-encoded proteins resolved the phylogenetic relationships among the main eustigmatophyte lineages and provided a framework for the interpretation of plastid gene gains and losses in the group. The ebo operon gain was inferred to have probably occurred within the order Eustigmatales, after the divergence of the two basalmost lineages (a newly discovered hitherto undescribed strain and the Pseudellipsoidion group). When looking for nuclear genes potentially compensating for plastid gene losses, we noticed a gene for a plastid-targeted acyl carrier protein that was apparently acquired by horizontal gene transfer from Phycorickettsia. The presence of this gene in all eustigmatophytes studied, including representatives of both principal clades (Eustigmatales and Goniochloridales), is a genetic footprint indicating that the eustigmatophyte-Phycorickettsia partnership started no later than in the last eustigmatophyte common ancestor.
- MeSH
- Biological Evolution * MeSH
- Genome, Plastid * MeSH
- Stramenopiles genetics microbiology MeSH
- Operon * MeSH
- Rickettsiaceae genetics MeSH
- Amino Acid Sequence MeSH
- Symbiosis MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The σI sigma factor from Bacillus subtilis is a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI Further analysis revealed that the majority of these genes were affected indirectly by σI The σI regulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σI in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organism B. subtilisIMPORTANCE In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σI regulon from the industrially important model Gram-positive bacterium Bacillus subtilis We reveal that σI affects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription machinery.
- MeSH
- Bacillus subtilis genetics MeSH
- Bacterial Proteins genetics metabolism MeSH
- DNA-Directed RNA Polymerases genetics MeSH
- Transcription, Genetic * MeSH
- Operon MeSH
- Promoter Regions, Genetic * MeSH
- Gene Expression Regulation, Bacterial * MeSH
- Regulon MeSH
- Sigma Factor genetics MeSH
- Transcriptome MeSH
- Iron metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Rickettsiales are obligate intracellular bacteria originally found in metazoans, but more recently recognized as widespread endosymbionts of various protists. One genus was detected also in several green algae, but reports on rickettsialean endosymbionts in other algal groups are lacking. Here we show that several distantly related eustigmatophytes (coccoid algae belonging to Ochrophyta, Stramenopiles) are infected by Candidatus Phycorickettsia gen. nov., a new member of the family Rickettsiaceae. The genome sequence of Ca. Phycorickettsia trachydisci sp. nov., an endosymbiont of Trachydiscus minutus CCALA 838, revealed genomic features (size, GC content, number of genes) typical for other Rickettsiales, but some unusual aspects of the gene content were noted. Specifically, Phycorickettsia lacks genes for several components of the respiration chain, haem biosynthesis pathway, or c-di-GMP-based signalling. On the other hand, it uniquely harbours a six-gene operon of enigmatic function that we recently reported from plastid genomes of two distantly related eustigmatophytes and from various non-rickettsialean bacteria. Strikingly, the eustigmatophyte operon is closely related to the one from Phycorickettsia, suggesting a gene transfer event between the endosymbiont and host lineages in early eustigmatophyte evolution. We hypothesize an important role of the operon in the physiology of Phycorickettsia infection and a long-term eustigmatophyte-Phycorickettsia coexistence.
The capacity for anoxygenic photosynthesis is scattered throughout the phylogeny of the Proteobacteria. Their photosynthesis genes are typically located in a so-called photosynthesis gene cluster (PGC). It is unclear (i) whether phototrophy is an ancestral trait that was frequently lost or (ii) whether it was acquired later by horizontal gene transfer. We investigated the evolution of phototrophy in 105 genome-sequenced Rhodobacteraceae and provide the first unequivocal evidence for the horizontal transfer of the PGC. The 33 concatenated core genes of the PGC formed a robust phylogenetic tree and the comparison with single-gene trees demonstrated the dominance of joint evolution. The PGC tree is, however, largely incongruent with the species tree and at least seven transfers of the PGC are required to reconcile both phylogenies. The origin of a derived branch containing the PGC of the model organism Rhodobacter capsulatus correlates with a diagnostic gene replacement of pufC by pufX. The PGC is located on plasmids in six of the analyzed genomes and its DnaA-like replication module was discovered at a conserved central position of the PGC. A scenario of plasmid-borne horizontal transfer of the PGC and its reintegration into the chromosome could explain the current distribution of phototrophy in Rhodobacteraceae.
- MeSH
- Photosynthesis * MeSH
- Phototrophic Processes MeSH
- Phylogeny MeSH
- Genome, Bacterial MeSH
- Evolution, Molecular * MeSH
- Multigene Family MeSH
- Operon MeSH
- Plasmids genetics metabolism MeSH
- Gene Transfer, Horizontal * MeSH
- DNA Replication MeSH
- Rhodobacteraceae classification genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.
- MeSH
- Agrobacterium enzymology genetics MeSH
- Genes, Bacterial MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Flavin Mononucleotide metabolism MeSH
- Catalytic Domain MeSH
- Kinetics MeSH
- Crystallography, X-Ray MeSH
- Protein Structure, Quaternary MeSH
- Models, Molecular MeSH
- NADPH Dehydrogenase chemistry genetics metabolism MeSH
- Operon MeSH
- Oxidative Stress MeSH
- Oxidoreductases chemistry genetics metabolism MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
ALKB-8 is a 2-oxoglutarate-dependent dioxygenase homologous to bacterial AlkB, which oxidatively demethylates DNA substrates. The mammalian AlkB family contains AlkB homologues denominated ALKBH1 to 8 and FTO. The C. elegans genome includes five AlkB-related genes, homologues of ALKBH1, 4, 6, 7, and 8, but lacks homologues of ALKBH2, 3, and 5 and FTO. ALKBH8 orthologues differ from other AlkB family members by possessing an additional methyltransferase module and an RNA binding N-terminal module. The ALKBH8 methyltransferase domain generates the wobble nucleoside 5-methoxycarbonylmethyluridine from its precursor 5-carboxymethyluridine and its (R)- and (S)-5-methoxycarbonylhydroxymethyluridine hydroxylated forms in tRNA Arg/UCG and tRNA Gly/UCC. The ALKBH8/ALKB-8 methyltransferase domain is highly similar to yeast TRM9, which selectively modulates translation of mRNAs enriched with AGA and GAA codons under both normal and stress conditions. In this report, we studied the role of alkb-8 in C. elegans. We show that downregulation of alkb-8 increases detection of lysosome-related organelles visualized by Nile red in vivo. Reversely, forced expression of alkb-8 strongly decreases the detection of this compartment. In addition, overexpression of alkb-8 applied in a pulse during the L1 larval stage increases the C. elegans lifespan.
- MeSH
- Caenorhabditis elegans embryology enzymology genetics MeSH
- Dioxygenases metabolism MeSH
- Longevity MeSH
- Down-Regulation genetics MeSH
- Embryo, Nonmammalian metabolism MeSH
- Animals, Genetically Modified MeSH
- Ketoglutaric Acids metabolism MeSH
- Larva metabolism MeSH
- Lysosomes metabolism MeSH
- Methyltransferases metabolism MeSH
- Operon MeSH
- Promoter Regions, Genetic MeSH
- Caenorhabditis elegans Proteins genetics metabolism MeSH
- RNA Interference MeSH
- S-Adenosylmethionine metabolism MeSH
- Aging metabolism MeSH
- Gene Expression Regulation, Developmental MeSH
- Green Fluorescent Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
A highly divergent 16S rRNA gene was found in one of the five ribosomal operons present in a species complex currently circumscribed as Scytonema hyalinum (Nostocales, Cyanobacteria) using clone libraries. If 16S rRNA sequence macroheterogeneity among ribosomal operons due to insertions, deletions or truncation is excluded, the sequence heterogeneity observed in S. hyalinum was the highest observed in any prokaryotic species thus far (7.3-9.0%). The secondary structure of the 16S rRNA molecules encoded by the two divergent operons was nearly identical, indicating possible functionality. The 23S rRNA gene was examined for a few strains in this complex, and it was also found to be highly divergent from the gene in Type 2 operons (8.7%), and likewise had nearly identical secondary structure between the Type 1 and Type 2 operons. Furthermore, the 16S-23S ITS showed marked differences consistent between operons among numerous strains. Both operons have promoter sequences that satisfy consensus requirements for functional prokaryotic transcription initiation. Horizontal gene transfer from another unknown heterocytous cyanobacterium is considered the most likely explanation for the origin of this molecule, but does not explain the ultimate origin of this sequence, which is very divergent from all 16S rRNA sequences found thus far in cyanobacteria. The divergent sequence is highly conserved among numerous strains of S. hyalinum, suggesting adaptive advantage and selective constraint of the divergent sequence.
- MeSH
- RNA, Bacterial chemistry genetics MeSH
- DNA, Bacterial genetics isolation & purification MeSH
- Phylogeny MeSH
- Nucleic Acid Conformation MeSH
- Operon * MeSH
- Promoter Regions, Genetic MeSH
- Ribosomes metabolism MeSH
- RNA, Ribosomal, 16S chemistry genetics MeSH
- Cyanobacteria classification genetics MeSH
- Publication type
- Journal Article MeSH
p-Cresol and indole are volatile biologically active products of the bacterial degradation of tyrosine and tryptophan respectively. They are typically produced by bacteria in animal intestines, soil and various sediments. Here, we demonstrate that the free-living eukaryote Mastigamoeba balamuthi and its pathogenic relative Entamoeba histolytica produce significant amounts of indole via tryptophanase activity. Unexpectedly, M. balamuthi also produces p-cresol in concentrations that are bacteriostatic to non-p-cresol-producing bacteria. The ability of M. balamuthi to produce p-cresol, which has not previously been observed in any eukaryotic microbe, was gained due to the lateral acquisition of a bacterial gene for 4-hydroxyphenylacetate decarboxylase (HPAD). In bacteria, the genes for HPAD and the S-adenosylmethionine-dependent activating enzyme (AE) are present in a common operon. In M. balamuthi, HPAD displays a unique fusion with the AE that suggests the operon-mediated transfer of genes from a bacterial donor. We also clarified that the tyrosine-to-4-hydroxyphenylacetate conversion proceeds via the Ehrlich pathway. The acquisition of the bacterial HPAD gene may provide M. balamuthi a competitive advantage over other microflora in its native habitat.
- MeSH
- Archamoebae genetics MeSH
- Bacteria genetics MeSH
- Genes, Bacterial * MeSH
- Indoles metabolism MeSH
- Carboxy-Lyases MeSH
- Cresols metabolism MeSH
- Operon MeSH
- Gene Transfer, Horizontal * MeSH
- S-Adenosylmethionine metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Trace amounts of the carcinogenic ethyl carbamate can appear in wine as a result of a reaction between ethanol and citrulline, which is produced from arginine degradation by some bacteria used in winemaking. In this study, arginine deiminase (ADI) pathway genes were evaluated in 44 Oenococcus oeni strains from wines originating from several locations in order to establish the relationship between the ability of a strain to degrade arginine and the presence of related genes. To detect the presence of arc genes of the ADI pathway in O. oeni, pairs of primers were designed to amplify arcA, arcB, arcC and arcD1 sequences. All strains contained these four genes. The same primers were used to confirm the organization of these genes in an arcABCD1 operon. Nevertheless, considerable variability in the ability to degrade arginine among these O. oeni strains was observed. Therefore, despite the presence of the arc genes in all strains, the expression patterns of individual genes must be strain dependent and influenced by the different wine conditions. Additionally, the presence of arc genes was also determined in the 57 sequenced strains of O. oeni available in GenBank, and the complete operon was found in 83% of strains derived from wine. The other strains were found to lack the arcB, arcC and arcD genes, but all contained sequences homologous to arcA, and some of them had also ADI activity.
An arsenate susceptibility test was performed with transformed and cultured Escherichia coli DH5α cells, which carried recombinant DNA of full-length arsenic (ars) operon, namely a putative membrane permease, ArsP; a transcriptional repressor, ArsR; an arsenate reductase, ArsC; and an arsenical-resistance membrane transporter, Acr3, from the Japanese urease-positive thermophilic Campylobacter lari (UPTC) CF89-12. The E. coli DH5α transformant showed reduced susceptibility to arsenate (~1536 μg/mL), compared to the control. Thus, these ars four-genes from the UPTC CF89-12 strain cells could confer a reduced susceptibility to arsenate in the transformed and E. coli DH5α cells. E. coli transformants with truncated ars operons, acr3 (acr3) and arsC-acr3 (∆arsC-acr3), of the ars operon, showed an MIC value of 384 μg/mL (~384 μg/mL), similar to the E. coli cells which carried the pGEM-T vector (control). Reverse transcription PCR confirmed in vivo transcription of recombinant full-length ars operon and deletion variants (∆acr3 and ∆arsC-acr3) in the transformed E. coli cells.
- MeSH
- Arsenic metabolism toxicity MeSH
- Campylobacter lari drug effects genetics metabolism MeSH
- Escherichia coli genetics metabolism MeSH
- Metabolic Networks and Pathways genetics MeSH
- Microbial Sensitivity Tests MeSH
- Operon * MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Gene Expression Profiling MeSH
- Transformation, Genetic MeSH
- Publication type
- Journal Article MeSH
