Response of Escherichia coli growth rate to osmotic shock

It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper- and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless “stored” growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan.Cell growth is the result of a complex system of biochemical processes and mechanical forces. For bacterial, plant, and fungal cells, growth requires both the synthesis of cytoplasmic components and the expansion of the cell wall, a stiff polymeric network that encloses these cells. It is well established that plant cells use turgor pressure, the outward normal force exerted by the cytoplasm on the cell wall, to drive mechanical expansion of the cell wall during growth (1, 2). In contrast, our understanding of the physical mechanisms of cell-wall expansion in bacteria is limited. Furthermore, the bacterial cell wall is distinct from its eukaryotic counterparts in both ultrastructure and chemical composition. In particular, the peptidoglycan cell wall of Gram-negative bacteria is extremely thin, comprising perhaps a molecular monolayer (3). This raises the question of whether these organisms require turgor pressure for cell-wall expansion, or whether they use a different strategy than organisms with thicker walls.Turgor pressure is established within cells according to the Morse equation, P = RT(C_in ? C_out), where C_in is the osmolarity of the cytoplasm, C_out is the osmolarity of the extracellular medium, R is the gas constant, and T is the temperature. In the Gram-negative bacterium Escherichia coli, P has been estimated to be 1–3 atm (4, 5). A primary role of the cell wall is to bear this load by balancing it with mechanical stress, thereby preventing cell lysis. In 1924, Walter proposed a theory of bacterial growth based on the premise that mechanical stress, in turn, is responsible for stretching the cell wall during growth (6). In support of this theory, he and others showed that the growth rate of a number of bacterial species, including E. coli, decreases as the osmolarity of their growth medium is increased (6–9) (Fig. 1A). This result would be predicted by the Morse equation if growth rate, e˙, scaled with pressure: e˙∼P∼−Cout. The decrease in growth rate did not depend on the chemical used to modulate the osmolarity, which demonstrated that this effect was not due to specific, toxic reactions. More recently, several theoretical studies have offered plausible molecular mechanisms by which turgor pressure could drive cell-wall expansion. These theories range from ones in which the cell wall is irreversibly stretched by turgor pressure when peptidoglycan cross-links are hydrolyzed (10, 11) to ones in which the rate of peptidoglycan biosynthesis is directly dependent on turgor pressure (12, 13). Two of these theories explicitly predict the scaling between cellular growth rate and pressure (12, 13), providing possible explanations of the classic experiments (6–9).Open in a separate windowFig. 1.E. coli cells maintain their elongation rate after hyperosmotic shock. (A) The population-averaged steady-state elongation rate as a function of the concentration of sorbitol in the growth medium, C_out (red circles). Also shown are the population-averaged elongation rates during phase III of recovery from a 400 mM hyperosmotic shock (blue triangles) and immediately after a 400 mM hypoosmotic shock (green squares). Each data point is averaged over 20–100 cells. Error bars indicate ±1 SE. (Inset) Definition of elongation rate. (B) E. coli stained with fluorescent WGA, imaged with phase and epifluorescence microscopy. (Lower) Schematic defining the length and width of a rod-shaped cell. (C) Longitudinal strain, ε_l, and radial strain, ε_w, as a function of medium osmolarity. Error bars indicate ±1 SE. (Inset) Tracked outlines of the wall of four cells before (red) and after (blue) plasmolysis. (D) (Left) Initial images of a single cell: phase, epifluorescence, and overlay. In the overlay, the epifluorescence kymograph is false-colored green and the inverse of the phase kymograph is false-colored red. (Right) Kymographs of this cell elongating and dividing during a 400 mM hyperosmotic shock. The kymographs consist of a montage of a one-pixel-wide region around the long axis of the cell, indicated by the dashed line. The white arrow indicates the time of the shock. The red arrow indicates the period of plasmolysis during which the cytoplasm is shorter than the cell wall. The black arrow indicates the formation of a septum during cell division. (E) The concentration of sorbitol in the growth medium versus time during a hyperosmotic shock. (F) The length of the cell walls of representative cells during the same shock. The background colors indicate the four phases of response. (G) The population-averaged elongation rate of both the cytoplasm and the cell wall during the same shock (n = 42 cells). The data were smoothed with a moving-average filter with a 2-min window for illustrative purposes. The blue dotted lines indicate the steady-state elongation rates in LB (upper line) and LB+400 mM sorbitol (lower line). The confidence intervals indicate ±1 SE. The cell wall could not be tracked indefinitely owing to dilution of the cell-wall stain. (Lower) Key describing the four phases of response to hyperosmotic shock.However, bacterial cells possess several mechanisms for regulating their cytoplasmic osmolarity in response to changes in their external osmotic environment (14). Specifically, E. coli imports and synthesizes compatible solutes, and imports ions, in response to high external osmolarities (15). For example, the concentration of potassium ions in the cytoplasm scales as the external osmolality (16). Therefore, it is unclear whether raising medium osmolarity actually causes a decrease in turgor pressure over long time scales. The inverse correlation between growth rate and medium osmolarity could result from another osmotic effect such as altered water potential within the cell, which could affect, for example, protein folding (17) or signaling (18). We sought to distinguish between these possibilities by measuring the elastic strain within the cell wall as a function of medium osmolarity and by determining the time scale over which osmotic shock (acute changes in medium osmolarity) modulates growth rate and cell-wall synthesis. If growth rate scales with turgor pressure, then (i) growth rate should also scale with cell-wall elastic strain, and therefore elastic strain should decrease with increasing medium osmolarity, and (ii) growth rate, and perhaps the rate of cell-wall synthesis, should rapidly change upon osmotic shock. However, if medium osmolarity modulates growth rate independently of pressure, these processes may adapt more slowly to changes in osmolarity.We found that elastic strain decreases only moderately with increasing medium osmolarity. By measuring the growth-rate response of E. coli across time scales that spanned four orders of magnitude we concluded that osmotic shock had little effect on growth rate, except in the case of plasmolysis (when P ≤ 0, causing the cytoplasm to separate from the cell wall). The growth rate of E. coli adapted slowly to changes in medium osmolarity, over the course of tens of minutes. Furthermore, when turgor pressure was restored after slight plasmolysis, E. coli cells quickly elongated to the length that they would have attained had they never been plasmolyzed. From this result, we inferred that peptidoglycan synthesis is insensitive to changes in turgor pressure. In support of this hypothesis, we found that the speed of MreB, a protein whose motion is dependent on peptidoglycan synthesis (19–21), is largely unaffected by osmotic shock. Therefore, our results demonstrate that cell-wall biosynthesis is the rate-limiting factor in cell-wall expansion and that turgor pressure is not required for cell-wall biosynthesis, but that pressure does play a simple role whereby it stretches recently assembled, unextended peptidoglycan.     

Pattern of microbial translocation in patients living with HIV-1 from Vietnam,Ethiopia and Sweden

Introduction

The role of microbial translocation (MT) in HIV patients living with HIV from low- and middle-income countries (LMICs) is not fully known. The aim of this study is to investigate and compare the patterns of MT in patients from Vietnam, Ethiopia and Sweden.

Methods

Cross-sectional samples were obtained from treatment-naïve patients living with HIV-1 and healthy controls from Vietnam (n=83; n=46), Ethiopia (n=9492; n=50) and Sweden (n=51; n=19). Longitudinal samples were obtained from a subset of the Vietnamese (n=24) in whom antiretroviral therapy (ART) and tuberculostatics were given. Plasma lipopolysaccharide (LPS), sCD14 and anti-flagellin IgG were determined by the endpoint chromogenic Limulus Amebocyte Assay and enzyme-linked immunosorbent assay.

Results

All three biomarkers were significantly increased in patients living with HIV-1 from all countries as compared to controls. No differences were found between males and females. Vietnamese and Ethiopian patients had significantly higher levels of anti-flagellin IgG and LPS, as compared to Swedes. ART reduced these levels for the Vietnamese. Vietnamese patients given tuberculostatics at initiation of ART had significantly lower levels of anti-flagellin IgG and higher sCD14. The biomarkers were lower in Vietnamese who did not develop opportunistic infection.

Conclusions

Higher MT is common in patients living with HIV compared to healthy individuals, and in patients from LMICs compared to patients from a high-income country. Treatment with tuberculostatics decreased MT while higher levels of MT are associated with a poorer clinical outcome.     

远红外线消毒柜对碗消毒效果的检测

为了解远红外线加热的消毒柜对餐具的消毒效果,用碗进行了试验观察。结果,加热至125℃作用10min,对碗上细菌繁殖体的杀灭率达90.0％以上,只能杀灭枯草杆菌黑色变种芽胞42.56％;装30只碗时,作用10min对自然菌的杀灭率可达90.5％,装40只碗时仅能杀灭64.9％。说明用该柜消毒餐具时装量不宜太多。     

Hypercapnic acidosis minimizes endotoxin-induced gut mucosal injury in rabbits

Objective  Recent evidence demonstrated that hypercapnic acidosis due to lung protective strategy was not only permissive but also even therapeutic for injured lung. Since the effects of hypercapnic acidosis on extra-pulmonary organs remain to be clarified, we tested the hypothesis that hypercapnic acidosis protects gut mucosal barrier function by modulating inflammation in a rabbit model of endotoxemia. Design  Prospective randomized animal study. Setting  University research laboratory. Subjects  Male New Zealand white rabbits. Interventions  Thirty-two animals were randomly allocated into two groups: normocapnia (n = 17) and hypercapnia (n = 15). The latter group received F_ICO₂ 5% under mechanical ventilation to achieve hypercapnia throughout the study periods, whereas the former with F_ICO₂ 0%. Measurements and results  Arterial blood gas, intramucosal pH (pHi) and portal blood flow were assessed at baseline, 2-h and 4-h infusion of lipopolysaccharide. At 4 h, ileal myeloperoxidase (MPO) activity and intestinal permeability were measured. The animals in the hypercapnia group showed apparent hypercapnic acidosis and progressive intramucosal acidosis at 4 h, accompanied by significantly lower intestinal permeability versus normocapnia group. Ileal MPO activity was comparable between the study groups. Conclusions  Hypercapnic acidosis attenuates endotoxin-induced gut barrier dysfunction possibly through neutrophil-independent mechanisms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

一次性使用卫生用品生产环境与产品的细菌污染调查

对上海5种卫生用品生产企业进行了细菌污染调查。产品细菌数低于允许标准的合格率为71.43％,车间空气的合格率为82.96％,工作台表面合格率为90.12％,工人手表面细菌数合格率为81.48％。原料、生产环节与工人手污染程度对细菌污染量有明显影响。     

多次涂抹法检测物体表面细菌总数

本实验采用棉拭子涂抹采样法在相同面积、相同部位的物体表面上作反复涂抹不同次数的采样,以比较不同涂抹次数对检出细菌总数的影响。结果发现,二次涂抹采样细菌总数明显多于一次涂抹,二者比较差别显著(P<0.01);而三次涂抹与二次相比,则无显著差异(P>0.05)。所以,表面涂抹采样至少反复二次,方可获得较高捕获率。     

Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation

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