Gram-negative bacteria balance synthesis of the outer membrane (OM), cell wall, and cytoplasmic contents during growth via unknown mechanisms. Here, we show that a dominant mutation (designated mlaA*, maintenance of lipid asymmetry) that alters MlaA, a lipoprotein that removes phospholipids from the outer leaflet of the OM of Escherichia coli, increases OM permeability, lipopolysaccharide levels, drug sensitivity, and cell death in stationary phase. Surprisingly, single-cell imaging revealed that death occurs after protracted loss of OM material through vesiculation and blebbing at celldivision sites and compensatory shrinkage of the inner membrane, eventually resulting in rupture and slow leakage of cytoplasmic contents. The death of mlaA* cells was linked to fatty acid depletion and was not affected by membrane depolarization, suggesting that lipids flow from the inner membrane to the OM in an energy-independent manner. Suppressor analysis suggested that the dominant mlaA* mutation activates phospholipase A, resulting in increased levels of lipopolysaccharide and OM vesiculation that ultimately undermine the integrity of the cell envelope by depleting the inner membrane of phospholipids. This novel cell-death pathway suggests that balanced synthesis across both membranes is key to the mechanical integrity of the Gram-negative cell envelope.

Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth. IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

Importance 43 44 How starved bacteria adapt to and multiply in replete nutrient conditions is intimately 45 linked to their history of previous growth, their physiological state, and the 46 surrounding environment. While automated equipment has enabled high-throughput 47 56 growth of most of the population, due to lipoteichoic-acid synthesis. These studies 57 highlight the challenges of accurate quantification of bacterial growth behaviors.58

A total of 38 samples, including 15 fish products, 11 meat products from retail shops and 12 pork products from a factory place, was examined for the presence of Yersiniaspecies. Six well‐known isolation protocols were used. A two‐stage enrichment procedure including 48 h incubation of sample homogenates in brain heart infusion at 8–10°C followed by incubation in sorbitol bile broth at 22–28°C for 48 h recovered the highest number ofYersiniaspp on cefsulodin–irgasan–Novobiocin agar. A total of 302 isolates, consisting mainly ofY intermedia(74, 41, 25),Y pestis(4, 8, 23),Y enterocolitica(5, 6, 5) andY pseudotuberculosis(0, 5, 1) was obtained from retail fish and meat products and from factory oriented pork products, respectively. Many of the isolates were not identified to species level because of their unusual biochemical reactions. None of theY enterocoliticaisolates contained the virulence plasmid as evidenced by their biotype (5 and 1) and inability to bind crystal violet.

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