Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches

Thiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for approximately 20% of global primary production. They have been predicted to contain TPP aptamers in the 3'UTR of some thiamine metabolism-related genes, but little is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine-related genes with putative TPP aptamers are not responsive to thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and the targeted mutation of the TPP aptamer in the HMP-P synthase (THIC) does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtSSSP is necessary for thiamine uptake and that PtTHIC is essential for thiamine biosynthesis. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

Functional exploration of the ubiquitous plasmid pEA29 reveals a novel signaling pathway that connects thiamine biosynthesis, bacterial respiration, and production of the exopolysaccharide amylovoran in Erwinia amylovora

Erwinia amylovora is a plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. This bacterium colonizes host vascular tissues via the production of exopolysaccharides (EPSs) including amylovoran. It is well established that the nearly ubiquitous plasmid pEA29 of E. amylovora is an essential virulence factor, but the underlying mechanism remains uncharacterized. Here, we demonstrated that pEA29 was required for E. amylovora to produce amylovoran and to form a biofilm, and this regulation was dependent on the thiamine biosynthesis operon thiOSGF. We then conducted carbohydrate and genetic analyses demonstrating that the thiamine-mediated effect on amylovoran production was indirect, as cells lacking thiOSGF produced an EPS that did not contain glucuronic acid, one of the key components of amylovoran, whereas the transcriptional activity and RNA levels of the amylovoran biosynthesis genes were not altered. Alternatively, addition of exogenous thiamine restored amylovoran production in the pEA29-cured strain of E. amylovora and positively impacted amylovoran production in a dose-dependent manner. Individual deletion of several chromosomal thiamine biosynthesis genes also affected amylovoran production, implying that a complete thiamine biosynthesis pathway is required for the thiamine-mediated effect on amylovoran production in E. amylovora. Finally, we determined that an imbalanced tricarboxylic acid cycle negatively affected amylovoran production, which was restored by addition of exogenous thiamine or overexpression of the thiOSGF operon. In summary, our report revealed a novel signaling pathway that impacts E. amylovora virulence in which thiamine biosynthesis enhances bacterial respiration that provides energetic requirements for the biosynthesis of EPS amylovoran.

Signatures of optimal codon usage predict metabolic ecology in budding yeasts

ABSTRACTReverse ecology is the inference of ecological information from patterns of genomic variation. One rich, heretofore underutilized, source of ecologically-relevant genomic information is codon optimality or adaptation. Bias toward codons that match the tRNA pool is robustly associated with high gene expression in diverse organisms, suggesting that codon optimization could be used in a reverse ecology framework to identify highly expressed, ecologically relevant genes. To test this hypothesis, we examined the relationship between optimal codon usage in the classic galactose metabolism (GAL) pathway and known ecological niches for 329 species of budding yeasts, a diverse subphylum of fungi. We find that optimal codon usage in the GAL pathway is positively correlated with quantitative growth on galactose, suggesting that GAL codon optimization reflects increased capacity to grow on galactose. Optimal codon usage in the GAL pathway is also positively correlated with human-associated ecological niches in yeasts of the CUG-Ser1 clade and with dairy-associated ecological niches in the family Saccharomycetaceae. For example, optimal codon usage of GAL genes is greater than 85% of all genes in the major human pathogen Candida albicans (CUG-Ser1 clade) and greater than 75% of genes in the dairy yeast Kluyveromyces lactis (family Saccharomycetaceae). We further find a correlation between optimization in the thiamine biosynthesis and GAL pathways. As a result, optimal codon usage in thiamine biosynthesis genes is also associated with dairy ecological niches in Saccharomycetaceae, which may reflect competition with co-occurring microbes for extracellular thiamine. This work highlights the potential of codon optimization as a tool for gaining insights into the metabolic ecology of microbial eukaryotes. Doing so may be especially illuminating for studying fungal dark matter—species that have yet to be cultured in the lab or have only been identified by genomic material.

The ThiL enzyme is a valid antibacterial target essential for both thiamine biosynthesis and salvage pathways in Pseudomonas aeruginosa

Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. Thiamine biosynthesis occurs in bacteria but not in humans; therefore, the enzymes in this pathway are attractive targets for antibiotic development. Among these enzymes, thiamine monophosphate kinase (ThiL) catalyzes the final step of this pathway, phosphorylating thiamine monophosphate to produce TPP. Here, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen responsible for hospital-acquired infections. We demonstrate that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability and results in growth defects of the ΔthiL strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the ΔthiL strain was markedly attenuated, compared with that of WT cells, with lower inflammatory cytokine induction and 103–104-fold decreased bacterial loads in an in vivo infection model in which the intracellular TPP level was in the submicromolar range. To validate P. aeruginosa ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a Vmax of 4.0 ± 0.2 nmol·min−1 and Km values of 111 ± 8 and 8.0 ± 3.5 μm for ATP and thiamine monophosphate, respectively. An in vitro small-molecule screening assay identified PaThiL inhibitors including WAY213613, a noncompetitive inhibitor with a Ki value of 13.4 ± 2.3 μm and potential antibacterial activity against P. aeruginosa. These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against P. aeruginosa.

The Iron Deficiency Response of Corynebacterium glutamicum and a Link to Thiamine Biosynthesis

ABSTRACT The response to iron limitation of the Gram-positive soil bacterium Corynebacterium glutamicum was analyzed with respect to secreted metabolites, the transcriptome, and the proteome. During growth in glucose minimal medium, iron limitation caused a shift from lactate to pyruvate as the major secreted organic acid complemented by l-alanine and 2-oxoglutarate. Transcriptome and proteome analyses revealed that a pronounced iron starvation response governed by the transcriptional regulators DtxR and RipA was detectable in the late, but not in the early, exponential-growth phase. A link between iron starvation and thiamine pyrophosphate (TPP) biosynthesis was uncovered by the strong upregulation of thiC. As phosphomethylpyrimidine synthase (ThiC) contains an iron-sulfur cluster, limiting activities of the TPP-dependent pyruvate–2-oxoglutarate dehydrogenase supercomplex probably cause the excretion of pyruvate and 2-oxoglutarate. In line with this explanation, thiamine supplementation could strongly diminish the secretion of these acids. The upregulation of thiC and other genes involved in thiamine biosynthesis and transport is presumably due to TPP riboswitches present at the 5′ end of the corresponding operons. The results obtained in this study provide new insights into iron homeostasis in C. glutamicum and demonstrate that the metabolic consequences of iron limitation can be due to the iron dependency of coenzyme biosynthesis. IMPORTANCE Iron is an essential element for most organisms but causes problems due to poor solubility under oxic conditions and due to toxicity by catalyzing the formation of reactive oxygen species (ROS). Therefore, bacteria have evolved complex regulatory networks for iron homeostasis aiming at a sufficient iron supply while minimizing ROS formation. In our study, the responses of the actinobacterium Corynebacterium glutamicum to iron limitation were analyzed, resulting in a detailed view on the processes involved in iron homeostasis in this model organism. In particular, we provide evidence that iron limitation causes TPP deficiency, presumably due to insufficient activity of the iron-dependent phosphomethylpyrimidine synthase (ThiC). TPP deficiency was deduced from the upregulation of genes controlled by a TPP riboswitch and secretion of metabolites caused by insufficient activity of the TPP-dependent enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. To our knowledge, the link between iron starvation and thiamine synthesis has not been elaborated previously.

ThiL is a valid antibacterial target that is essential for both thiamine biosynthesis and salvage pathway in Pseudomonas aeruginosa

ABSTRACTThiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. As thiamine biosynthesis occurs in bacteria but not humans, bacterial thiamine biosynthesis is an attractive target for antibiotic development. Among enzymes in the thiamine biosynthetic pathway, thiamine monophosphate kinase (ThiL) catalyzes the final step of the pathway, phosphorylating thiamine monophosphate (TMP) to produce TPP. In this work, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen of hospital-acquired infections. We demonstrated that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability, showing growth defects of the ΔthiL mutant even in the presence of thiamine derivatives except TPP. Most importantly, the pathogenesis of the ΔthiL mutant was markedly attenuated compared to wild-type bacteria, with lower inflammatory cytokine induction and 103~104 times decreased bacterial load in an in vivo infection model where the intracellular TPP level is in the submicromolar range. In order to validate P. aeruginosa ThiL (PaThiL) as a new drug target, we further characterized its biochemical properties determining a Vmax of 4.0±0.2 nomol·min−1 and KM values of 111±8 and 8.0±3.5μM for ATP and TMP, respectively. A subsequent in vitro small molecule screening identified PaThiL inhibitors including WAY213613 that is a noncompetitive inhibitor with a Ki value of 13.4±2.3 μM and a potential antibacterial activity against P. aeruginosa. This study proved that PaThiL is a new drug target against P. aeruginosa providing comprehensive biological and biochemical data that could facilitate to develop a new repertoire of antibiotics.

Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity

Verticillium dahliae (V. dahliae) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops.

Thiamine pyrophosphate riboswitches in Bacteroides species regulate transcription or translation of thiamine transport and biosynthesis genes

Thiamine (vitamin B1) and its phosphorylated precursors are necessary for decarboxylation reactions required in carbohydrate and branched chain amino acid metabolism. Due to its critical roles in central metabolism, thiamine is essential for human and animal hosts and their resident gut microbes. However, little is known about how thiamine availability shapes the composition of gut microbial communities and the physiology of individual species within those communities. Our previous work has implicated both thiamine biosynthesis and transport activities in the fitness of Bacteroides species. To better understand thiamine-dependent gene regulation in Bacteroides, we examined thiamine biosynthesis and transport genes in three representative species: Bacteroides thetaiotaomicron, Bacteroides uniformis, and Bacteroides vulgatus. All three species possess thiamine biosynthetic operons controlled by highly conserved cis-acting thiamine pyrophosphate (TPP) riboswitches. B. thetaiotaomicron and B. uniformis have additional TPP riboswitch-controlled operons encoding thiamine transport functions. Transcriptome analyses showed that each Bacteroides species had a distinct transcriptional response to exogenous thiamine. Analysis of transcript levels and translational fusions demonstrated that in B. thetaiotaomicron, the TPP riboswitch upstream of biosynthesis genes acts at the level of transcription, while TPP riboswitches upstream of transport operons work at the level of translation. In B. uniformis and B. vulgatus, TPP riboswitches work at the transcriptional level to control downstream operons. The varying responses to exogenous thiamine and use of varied regulatory mechanisms may play an important role in niche establishment by the Bacteroidetes in the complex and constantly shifting gut environment.ImportanceBacteroides species are important and abundant members of human gut microbiome communities. Their activities in the gut are influenced by constant changes in nutrient availability. In this study, we investigated the genetic basis of thiamine (Vitamin B1) uptake and biosynthesis in three representative Bacteroides species. We found species-specific differences in the response to exogenous thiamine, and distinct mechanisms for regulation of uptake and biosynthesis gene expression. Our work implies that gut Bacteroides have evolved distinct strategies for making or acquiring an essential nutrient. These mechanisms may play an important role in the success of Bacteroides in establishing a niche within complex gut microbiome communities.