Fig 2: B. subtilis Bcd functionally substitutes for the loss of GudB in S. aureus, which lacks a Bcd homolog.a Phylogenetic tree shows that Bcd and GudB/RocG form two monophyletic clades. Multiple Bcd and GudB/RocG paralogs can be found in a single species, e.g., H. halodurans, which are color-coded accordingly. b Taxonomic distribution of Bcd homologs. The pie-chart size and black section represent the number of bacterial genomes and the frequency of Bcd presence, respectively. Terrabacteria mostly include Gram-positive bacteria such as Bacillota (Firmicutes); FCB phylum consists of Fibrobacteriota, Chloraobiota, and Bacteriodota. Less well-defined superphyla are loosely categorized by color. c Structural superposition of B. subtilis GudB1 structure (PDB 3k8z) with predicted structures of B. subtilis Bcd and S. aureus GudB reveals high resemblance among these GDHs. d Plasmid borne-Bcd partially restores GDH activity in a S. aureus ∆gudB mutant. EV, empty vector. Statistical significance was determined using Student’s t-test. *P < 0.05, ***P < 0.001, ns = not significant. e Growth defects of the S. aureus ∆gudB mutant are fully complemented by WT Bcd and its cognate GudB but not by the Bcd(K80A) mutant. Student’s t-test (n = 4), ***P < 0.001. f Western blot analysis shows endogenous GudB and plasmid-borne GudB expression in S. aureus. Anti-GudB does not cross-react with Bcd despite structural similarity. An asterisk indicates non-specific band.

Fig 3: Glutamate homeostasis is essential for biofilm development in B. subtilis.a Inactivation of the major GDH (GudB) and Bcd abolishes the wrinkled morphology of biofilm colonies. Removing gudB, rocG and bcd together results in aberrant biofilm expansion. (Left) A schematic of glutamate metabolism shows that biomass production requires external glutamate and ammonium generated internally via GDH-mediated glutamate degradation. b Pellicle biofilms at the air-liquid interface lose their rugose structures in the absence of GudB and Bcd. These defects are exacerbated in the ΔgudB∆rocG∆bcd triple mutant. Chromosomal expression of bcd from the sacA locus under its native promoter provides varying degrees of complementation in different mutants.

Fig 4: Bcd exhibits NAD+-dependent GDH activity in wild-type (WT) B. subtilis, while RocA shows NAD(P)+-dependent GDH activity only in the absence of GudB and RocG.a Simplified diagram of nitrogen and carbon metabolism in S. aureus and B. subtilis. The glutamate synthases GltA-GltB convert α-ketoglutarate (αKG) and glutamine into two molecules of glutamate. The glutamine synthetase GlnA catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. GudB and RocG deaminate glutamate to produce αKG, which feeds into the tricarboxylic acid cycle (TCA), generating acetyl coenzyme A (acetyl-CoA) through pyruvate oxidation. B. subtilis carries both GudB and RocG, while S. aureus harbors only GudB. Additional glutamate degrading enzymes (indicated by question marks) remain unidentified. b B. subtilis produces two additional GDHs of unknown identity, as revealed by in-gel activity staining. Independent double knockouts (∆gudB∆rocG and ∆rocG∆gudB) show identical results. c Denaturing PAGE-immunoblot using the B. subtilis GudB antibody confirms proper gudB expression in the GudB-proficient strains. d Genetic knockouts confirm Bcd and RocA contributions to GDH activity. Both enzymes use NAD+ as the coenzyme (left) but only RocA utilizes NADP+ as the electron acceptor (right). e Zymogram analysis of a single bcd mutant and bcd-complemented strain rules out secondary mutations, substantiating Bcd as an alternative GDH. Zymography using NAD+ and glutamate substrate is shown. GDH activity of GudB and RocG was not detected in this assay, possibly due to the sequestration of GudB by GltAB and low transcription of rocG in LB and MSgg media.

Fig 5: Activity of enzymes that support ATP levels in the retina. A: Creatine kinase (CK) activity. B: Lactate dehydrogenase (LDH) activity. C: Aspartate aminotransferase (AST) activity. D: Glutamate dehydrogenase (GDH) activity. The activities of these enzymes were unaffected under all the experimental conditions. One-way analysis of variance (ANOVA) showed no statistically significant changes ( p value less than 0.05). Values represent mean ± standard error of the mean (n = 6–8 for each group). Abbreviations: C, control; Cyt, proinflammatory cytokines; HG, high glucose; HG + Cyt, high glucose + proinflammatory cytokines.