Getting the Payload in Place - Unravelling the Complexities of Making a Bacterial Microcompartment.

Bacterial microcompartments (BMCs) are complex macromolecular assemblies composed of any outer protein shell that encases a specific metabolic pathway cargo. Recent research is now starting to unravel some of the processes that are involved in directing the enzyme cargo to the inside of the BMC. In particular, an article in this issue of J Bacteriol by N. W. Kennedy, C. E. Mills, C. H. Abrahamson, A. Archer, et al. (J Bacteriol 204:e00576-21, 2022, https://doi.org/10.1128/jb.00576-21) highlights the role played by the shell protein PduB in coordinating this internalization process.

Pyridoxal and α-Ketoglutarate Independently Improve Function of Saccharomyces cerevisiae Thi5 in the Metabolic Network of Salmonella enterica.

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 (ScThi5) required modification of the underlying metabolic network, while LpThi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of ScThi5 and report strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and ScThi5 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 ScThi5 function in S. enterica. Finally, directly increasing αKG and PLP with supplementation improved function of both ScThi5 and relevant variants of Thi5 from Legionella pneumophila (LpThi5). The data herein suggest structural differences between ScThi5 and LpThi5 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 ScThi5 and LpThi5 contributes to their functional differences in a Salmonella enterica host.

Adaptive Synthesis of a Rough Lipopolysaccharide in Geobacter sulfurreducens for Metal Reduction and Detoxification.

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 expressed only 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 (ΔrfaQ) or complete (ΔrfaC) 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 surface 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 extracellular mineralization of the toxic uranyl cation but were unable to prevent 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 an LPS without the repeating sugars (O-antigen) that decorate the surface of most Gram-negative bacteria, but the biological significance of this adaptive feature was not previously 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.

Functional characterization of the HMP-P synthase of Legionella pneumophila (Lpg1565).

The production of the pyrimidine moiety in thiamine synthesis, 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P), has been described to proceed through the Thi5-dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP-P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila. Unlike ScThi5, LpThi5 functioned in vivo in Salmonella enterica under multiple growth conditions. The protein LpThi5 is a dimer that binds pyridoxal-5'-phosphate (PLP), apparently without a solvent-exposed Schiff base. A small percentage of LpThi5 protein co-purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function of LpThi5. IMPORTANCE: Thiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe-S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5-dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5-dependent pathway is necessary for thiamine biosynthesis in Legionella pneumophila and provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function.

SNZ3 Encodes a PLP Synthase Involved in Thiamine Synthesis in Saccharomyces cerevisiae.

Pyridoxal 5'-phosphate (the active form of vitamin B6) 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.

Induction of the Sugar-Phosphate Stress Response Allows Saccharomyces cerevisiae 2-Methyl-4-Amino-5-Hydroxymethylpyrimidine Phosphate Synthase To Function in Salmonella enterica.

UNLABELLED: 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. IMPORTANCE: This 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.