Integrated biphasic growth rate, gene expression, 1 and cell-size homeostasis behaviour of single B. 2 subtilis cells 3 Niclas Nordholt 1,2+ 7 2 Current affiliation: Bundesanstalt für Materialforschung und 8 -prüfung, Running title: Biphasic growth behaviour of single B. subtilis cells 12 1 Biphasic growth behaviour of single B. subtilis cells ABSTRACT 13The growth rate of single bacterial cells is continuously disturbed by random fluctu-14 ations in biosynthesis rates and by deterministic cell-cycle events, such as division, 15 genome duplication, and septum formation. It is not understood whether, and 16 how, bacteria reject these disturbances. Here we quantified growth and consti-17 tutive protein expression dynamics of single Bacillus subtilis cells, as a function 18 of cell-cycle-progression. Variation in birth size and growth rate, resulting from 19 unequal cell division, is largely compensated for when cells divide again. We anal-20 ysed the cell-cycle-dynamics of these compensations and found that both growth 21 and protein expression exhibited biphasic behaviour. During a first phase of vari-22 able duration, the absolute rates were approximately constant and cells behaved 23 as sizers. In the second phase, rates increased and growth behaviour exhibited 24 characteristics of a timer-strategy. This work shows how cell-cycle-dependent rate 25 adjustments of biosynthesis and growth are integrated to compensate for physio-26 logical disturbances caused by cell division. 27 28 IMPORTANCE 29 Under constant conditions, bacterial populations can maintain a fixed average 30 cell size and constant exponential growth rate. At the single cell-level, however, 31 cell-division can cause significant physiological perturbations, requiring compen-32 satory mechanisms to restore the growth-related characteristics of individual cells 33 toward that of the average cell. Currently, there is still a major gap in our under-34 standing of the dynamics of these mechanisms, i.e. how adjustments in growth, 35 metabolism and biosynthesis are integrated during the bacterial cell-cycle to com-36 pensate the disturbances caused by cell division. Here we quantify growth and 37 constitutive protein expression in individual bacterial cells at sub-cell-cycle reso-38 lution. Significantly, both growth and protein production rates display structured 39 and coordinated cell-cycle-dependent dynamics. These patterns reveal the dy-40 namics of growth rate and size compensations during cell-cycle progression. Our 41 findings provide a dynamic cell-cycle perspective that offers novel avenues for the 42 interpretation of physiological processes that underlie cellular homeostasis in bac-43 teria. 44 Under constant conditions, isogenic populations of bacteria maintain time-invariant 46 distributions of cell size, generation time and macromolecular composition. This 47growth mode is called balanced growth 1,2,3,4 . Yet, individual cells display molec-48 ular fluctuations that are the source for non-genetic heterogeneity within an iso-49 geni...