Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis.

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single-molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

Mechanisms of growth and patterns of gene expression in oxygen-deprived rice coleoptiles.

Coleoptiles of rice (Oryza sativa) seedlings grown under water commonly elongate by up to 1 mm h(-1) to reach the atmosphere. We initially analysed this highly specialized phenomenon by measuring epidermal cell lengths along the coleoptile axis to determine elongation rates. This revealed a cohort of cells in the basal zone that elongated rapidly following emergence from the embryo, reaching 200 µm within 12 h. After filming coleoptiles in vivo for a day, kinematic analysis was applied. Eight time-sliced 'segments' were defined by their emergence from the embryo at four-hourly intervals, revealing a mathematically simple growth model. Each segment entering the coleoptile from the embryo elongated at a constant velocity, resulting in accelerating growth for the entire organ. Consistent with the epidermal cell lengths, relative rates of elongation (mm mm(-1) h(-1)) were tenfold greater in the small, newly emerged basal segments than the older distal tip segments. This steep axial gradient defined two contrasting growth zones (bases versus tips) in which we measured ATP production and protein, RNA and DNA content, and analysed the global transcriptome under steady-state normoxia, hypoxia (3% O2) and anoxia. Determination of the transcriptome revealed tip-specific induction of genes encoding TCP [Teosinte Branched1 (Tb1) of maize, Cycloidea (Cyc), and Proliferating Cell Factor (Pcf)] transcription factors, RNA helicases, ribosomal proteins and proteins involved in protein folding, whilst expression of F-box domain-containing proteins in the ubiquitin E3-SCF complex (Skp, Cullin, F-box containing complex) was induced specifically in bases under low oxygen conditions. We ascribed the sustained elongation under hypoxia to hypoxia-specific responses such as controlled suppression of photosystem components and induction of RNA binding/splicing functions, indicating preferential allocation of energy to cell extension.

Quantifying ATP turnover in anoxic coleoptiles of rice (Oryza sativa) demonstrates preferential allocation of energy to protein synthesis.

Oxygen deprivation limits the energy available for cellular processes and yet no comprehensive ATP budget has been reported for any plant species under O(2) deprivation, including Oryza sativa. Using 3-d-old coleoptiles of a cultivar of O. sativa tolerant to flooding at germination, (i) rates of ATP regeneration in coleoptiles grown under normoxia (aerated solution), hypoxia (3% O(2)), and anoxia (N(2)) and (ii) rates of synthesis of proteins, lipids, nucleic acids, and cell walls, as well as K(+) transport, were determined. Based on published bioenergetics data, the cost of synthesizing each class of polymer and the proportion of available ATP allocated to each process were then compared. Protein synthesis consumed the largest proportion of ATP synthesized under all three oxygen regimes, with the proportion of ATP allocated to protein synthesis in anoxia (52%) more than double that in normoxic coleoptiles (19%). Energy allocation to cell wall synthesis was undiminished in hypoxia, consistent with preferential elongation typical of submerged coleoptiles. Lipid synthesis was also conserved strongly in O(2) deficits, suggesting that membrane integrity was maintained under anoxia, thus allowing K(+) to be retained within coleoptile cells. Rates of protein synthesis in coleoptiles from rice cultivars with contrasting tolerance to oxygen deficits (including mutants deficient in fermentative enzymes) confirmed that synthesis and turnover of proteins always accounted for most of the ATP consumed under anoxia. It is concluded that successful establishment of rice seedlings under water is largely due to the capacity of coleoptiles to allocate energy to vital processes, particularly protein synthesis.