Pyridoxal 5′-phosphate (the active form of vitamin B 6 ) is a cofactor that is important for a broad number of biochemical reactions and is essential for all forms of life. Organisms that can synthesize pyridoxal 5′-phosphate use either the deoxyxylulose phosphate-dependent or -independent pathway, the latter is encoded by a two-component pyridoxal 5′-phosphate synthase. Saccharomyces cerevisiae contains three paralogs of the two-component SNZ/SNO pyridoxal 5′-phosphate synthase. Past work identified the biochemical activity of Snz1p , Sno1p and provided in vivo data that SNZ1 was involved in pyridoxal 5′-phosphate biosynthesis. Snz2p and Snz3p were considered redundant isozymes and no growth condition requiring their activity was reported. Genetic data herein showed that either SNZ2 or SNZ3 are required for efficient thiamine biosynthesis in Saccharomyces cerevisiae . Further, SNZ2 or SNZ3 alone could satisfy the cellular requirement for pyridoxal 5′-phosphate (and thiamine), while SNZ1 was sufficient for pyridoxal 5′-phosphate synthesis only if thiamine was provided. qRT-PCR analysis determined that SNZ2 , 3 are repressed ten-fold by the presence thiamine. In total, the data were consistent with a requirement for PLP in thiamine synthesis, perhaps in the Thi5p enzyme, that could only be satisfied by SNZ2 or SNZ3 . Additional data showed that Snz3p is a pyridoxal 5′-phosphate synthase in vitro and is sufficient to satisfy the pyridoxal 5′-phosphate requirement in Salmonella enterica when the medium has excess ammonia.

The ability of some metal-reducing bacteria to produce a rough (no O-antigen) lipopolysaccharide (LPS) could facilitate surface interactions with minerals and metal reduction. Consistent with this, the laboratory model metal reducer Geobacter sulfurreducens PCA produced two rough LPS isoforms (with or without a terminal methyl-quinovosamine sugar) when growing with the soluble electron acceptor, fumarate, but only expressed the shorter and more hydrophilic variant when reducing iron oxides. We reconstructed from genomic data conserved pathways for the synthesis of the rough LPS and generated heptosyltransferase mutants with partial (Δ rfaQ ) and complete (Δ rfaC ) truncations in the core oligosaccharide. The stepwise removal of the LPS core sugars reduced the hydrophilicity of the cell and increased outer membrane vesiculation. These changes in outer membrane charge and remodeling did not substantially impact planktonic growth but disrupted the developmental stages and structure of electroactive biofilms. Furthermore, the mutants assembled conductive pili for the extracellular mineralization of the toxic uranyl cation, yet were unable to prevent the permeation and mineralization of the radionuclide in the cell envelope. Hence, not only does the rough LPS promote cell-cell and cell-mineral interactions critical to biofilm formation and metal respiration, but it also functions as a permeability barrier to toxic metal cations. In doing so, the rough LPS maximizes the extracellular reduction of soluble and insoluble metals and preserves cell envelope functions critical to the environmental survival of Geobacter bacteria in metal rich environments and their performance in bioremediation and bioenergy applications. Importance Some metal-reducing bacteria produce a lipopolysaccharide (LPS) without the repeating sugars (O-antigen) that decorate the surface of most Gram-negative bacteria, but the biological significance of this adaptive feature has never been investigated. Using the model representative Geobacter sulfurreducens strain PCA and mutants carrying stepwise truncations in the LPS core sugars, we demonstrate the importance of the rough LPS in the control of cell surface chemistry during the respiration of iron minerals and the formation of electroactive biofilms. Importantly, we describe hitherto overlooked roles for the rough LPS in metal sequestration and outer membrane vesiculation that are critical for the extracellular reduction and detoxification of toxic metals and radionuclides. These results are of interest for the optimization of bioremediation schemes and electricity-harvesting platforms using these bacteria.

Thiamine pyrophosphate is a required cofactor for all forms of life. The pyrimidine moiety of thiamine, 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P), is synthesized by different mechanisms in bacteria and plants compared to fungi. In this study, Salmonella enterica was used as a host to probe requirements for activity of the yeast HMP-P synthase, Thi5p. Thi5p synthesizes HMP-P from histidine and pyridoxal-5-phosphate and was reported to use a backbone histidine as the substrate, which would mean that it was a single-turnover enzyme. Heterologous expression of Thi5p did not complement an S. enterica HMP-P auxotroph during growth with glucose as the sole carbon source. Genetic analyses described here showed that Thi5p was activated in S. enterica by alleles of sgrR that induced the sugar-phosphate stress response. Deletion of ptsG (encodes enzyme IICB [EIICB] of the phosphotransferase system [PTS]) also allowed function of Thi5p and required sgrR but not sgrS. This result suggested that the role of sgrS in activation of Thi5p was to decrease PtsG activity. In total, the data herein supported the hypothesis that one mechanism to activate Thi5p in S. enterica grown on minimal medium containing glucose (minimal glucose medium) required decreased PtsG activity and an unidentified gene regulated by SgrR. IMPORTANCEThis work describes a metabolic link between the sugar-phosphate stress response and the yeast thiamine biosynthetic enzyme Thi5p when heterologously expressed in Salmonella enterica during growth on minimal glucose medium. Suppressor analysis (i) identified a mutant class of the regulator SgrR that activate sugar-phosphate stress response constitutively and (ii) determined that Thi5p is conditionally active in S. enterica. These results emphasized the power of genetic systems in model organisms to uncover enzyme function and underlying metabolic network structure.T hiamine pyrophosphate (TPP) is a cofactor for many central metabolic enzymes and is required at low levels by all organisms. Humans require dietary intake of thiamine, which is biosynthesized by a variety of plants, bacteria, and fungi. TPP is composed of two independently synthesized moieties, 5-(2-hydroxyethyl)-4-methylthiazole phosphate (THZ-P) and 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P). In bacteria and plants, the first steps of the HMP-P biosynthesis pathway are shared with purine biosynthesis (Fig. 1) (1-3). In these organisms, the radical S-adenosylmethionine (SAM) enzyme ThiC catalyzes an intramolecular rearrangement of the purine intermediate 5-aminoimidazole ribotide (AIR) to the pyrimidine HMP-P (4, 5).In contrast, fungi do not contain a ThiC homolog; under aerobic conditions, HMP-P is synthesized by the Thi5p enzyme family. In vivo labeling in yeast implicated histidine and pyridoxine as precursors to HMP-P biosynthesis under aerobic conditions ( , and other species have between zero and five copies of this gene (9). Genetic analysis in S. cerevisiae demonstrated that the four enzymes are f...

Thiamine pyrophosphate (TPP), the active form of Vitamin B 1 , is a cofactor important for several enzymes in central metabolism including transketolase, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase. The cofactor is comprised of two moieties, 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate (HMP-PP) and 4-methyl-5-β-hydroxyethylthiazole phosphate (THZ-P), that are independently synthesized and combined to form TPP (Jurgenson et al., 2009). The pyrimidine precursor HMP-P is synthesized from an intermediate in the purine biosynthetic pathway (aminoimidazole ribotide) by the phosphomethylpyrimidine synthase ThiC (E.C. 4.1.99.17) an enzyme encoded in plants, archaea, and most bacteria (Jurgenson et al., 2009). However, some organisms, notably Saccharomyces cerevisiae, use Thi5 (HMP-P synthase) in place of ThiC to generate HMP-P. Labeling studies showed that the atoms of

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica . Function of Thi5 from Saccharomyces cerevisiae ( Sc Thi5) required modification of the underlying metabolic network, while Lp Thi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of Sc Thi5 and report a strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and Sc Thi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved Sc Thi5 function in S. enterica . Finally, directly increasing αKG and PLP with supplementation improved function of both Sc Thi5 and relevant variants of Thi5 from Legionella pneumophila ( Lp Thi5). The data herein suggest structural differences between Sc Thi5 and Lp Thi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica . IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between Sc Thi5 and Lp Thi5 contribute to their functional differences in a Salmonella enterica host.