Poly(3-hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation.

Numerous bacteria accumulate poly(3-hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species.

Increased acid resistance of the archaeon, Metallosphaera sedula by adaptive laboratory evolution.

Extremely thermoacidophilic members of the Archaea such as the lithoautotroph, Metallosphaera sedula, are among the most acid resistant forms of life and are of great relevance in bioleaching. Here, adaptive laboratory evolution was used to enhance the acid resistance of this organism while genomics and transcriptomics were used in an effort to understand the molecular basis for this trait. Unlike the parental strain, the evolved derivative, M. sedula SARC-M1, grew well at pH of 0.90. Enargite (Cu3AsS4) bioleaching conducted at pH 1.20 demonstrated SARC-M1 leached 23.78 % more copper relative to the parental strain. Genome re-sequencing identified two mutations in SARC-M1 including a nonsynonymous mutation in Msed_0408 (an amino acid permease) and a deletion in pseudogene Msed_1517. Transcriptomic studies by RNA-seq of wild type and evolved strains at various low pH values demonstrated there was enhanced expression of genes in M. sedula SARC-M1 encoding membrane complexes and enzymes that extrude protons or that catalyze proton-consuming reactions. In addition, M. sedula SARC-M1 exhibited reduced expression of genes encoding enzymes that catalyze proton-generating reactions. These unique genomic and transcriptomic features support a model for increased acid resistance arising from enhanced control over cytoplasmic pH.

Role of an archaeal PitA transporter in the copper and arsenic resistance of Metallosphaera sedula, an extreme thermoacidophile.

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching.