The transition from unicellular to multicellular growth requires diversification of cellular functions within genetically identical populations. In Bacillus subtilis, biofilm formation is historically viewed as a developmental precursor to sporulation along a linear pathway. Here, we show that biofilm formation and sporulation instead diverge along a branched pathway. A subpopulation that first initiates sporulation catabolizes lipoteichoic acid through the sequential action of the enzymes ShfP (Sporulation heterogeneity factor Poison) and PhoA (alkaline phosphatase A), leading to the release of millimolar concentrations of glycerol. This glycerol impedes sporulation by disrupting cell wall synthesis and cytoplasmic pH, necessitating counteraction by another protein, ShfA (Sporulation heterogeneity factor Antidote). The extracellular glycerol, however, acts as a morphogen that directs neighboring cells to initiate biofilm formation, which we directly visualize in developing populations of cells. Thus, B. subtilis multicellularity emerges through a branched developmental program in which sporulating cells generate the cue that creates the biofilm-producing lineage via cell-cell communication through repurposing of a canonical intracellular metabolite.
Taylor B Updegrove, V. Pande, Fukang She et al.· Science Advances· 0 citations
Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.
R. Wilkes, P. Suthers, A. Borchert et al.· bioRxiv· 3 citations
