Contrasting Factors on the Kinetic Path to Protein Complex Formation Diminish the Effects of Crowding Agents

AbstractThe crowded environment of cells poses a challenge for rapid protein-protein association. Yet, it has been established that the rates of association are similar in crowded and in dilute solutions. Here we probe the pathway leading to fast association between TEM1 β-lactamase and its inhibitor protein BLIP in crowded solutions. We show that the affinity of the encounter complex, the rate of final complex formation, and the structure of the transition state are similar in crowded solutions and in buffer. The experimental results were reproduced by calculations based on the transient-complex theory for protein association. Both experiments and calculations suggest that while crowding agents decrease the diffusion constant of the associating proteins, they also induce an effective excluded-volume attraction between them. The combination of the two opposing effects thus results in nearly identical overall association rates in diluted and crowded solutions

Food selectivity and diet switch can explain the slow feeding of herbivorous coral-reef fishes during the morning.

Most herbivorous coral-reef fishes feed slower in the morning than in the afternoon. Given the typical scarcity of algae in coral reefs, this behavior seems maladaptive. Here we suggest that the fishes' slow feeding during the morning is an outcome of highly selective feeding on scarcely found green algae. The rarity of the food requires longer search time and extended swimming tracks, resulting in lower bite rates. According to our findings by noon the fish seem to stop their search and switch to indiscriminative consumption of benthic algae, resulting in apparent higher feeding rates. The abundance of the rare preferable algae gradually declines from morning to noon and seems to reach its lowest levels around the switch time. Using in situ experiments we found that the feeding pattern is flexible, with the fish exhibiting fast feeding rates when presented with ample supply of preferable algae, regardless of the time of day. Analyses of the fish's esophagus content corroborated our conclusion that their feeding was highly selective in the morning and non-selective in the afternoon. Modeling of the fishes' behavior predicted that the fish should perform a diel diet shift when the preferred food is relatively rare, a situation common in most coral reefs found in a warm, oligotrophic ocean

The fish selectively feed on <i>Ulva</i> sp. in the morning hours.

<p><b>A</b> Average (±SE) ratio between green algae (Chlorophytes) and the red alga <i>J. rubens</i> (Jr) in the esophagi of <i>A. nigrofuscus</i> (diet) and on ambient rocks at 10:00 (am) and 17:00 (pm). The rocks were collected at the same time and site where the fish were captured at the shallow reef (*** - P<0.005; NS – non significant; Tukey Post hoc). <b>B</b> Average (±SE) of consumed algae (dry weight) in the esophagi of <i>A. nigrofuscus</i>.</p

The abundance of <i>Ulva</i> sp. at areas exposed to grazing decreases only at morning hours.

<p>Average change (±SE) in the abundance of <i>Ulva</i> sp. (number of “spots” per 10×10 cm plate) of <i>Ulva</i> sp. on non-manipulated and caged plates between 7:00 to 12:00 (am, open bars) and between 12:00 to 17:00 (pm, full bars).</p

The fish swim longer distances and exhibit lower biting rates at early day hours.

<p>Average length (±SE) of swimming tracks (full line) and number of bites (dashes line) exhibited by <b>A</b> Z. <i>xanthurum</i> and <b>B </b><i>A. nigrofuscus</i> during 5 min intervals in different times of the day at the shallow reef (<3 m). Post hoc analysis (Tukey) indicated that the data points marked on the figure with the same letters were not significantly different (<i>P</i>>0.9), whereas the differences between those marked with different letters were highly significant (<i>P</i><0.001).</p

Model results: alternating between selective and non-selective feeding seems to be a good strategy for a wide range of conditions.

<p>The horizontal axis is taken as the overall edible algae frequency in the fish's habitat (), and the vertical axis is taken as the relative frequency of a highly nutritious alga amongst all algae verities . White areas define parameter ranges under which a switch from selective feeding to bulk feeding is favorable even when diminishing benefit is not assumed (); gray areas are the additional parameter ranges under which a switch between strategies is favorable when adding a diminishing benefit function to the nutritional value of the rare, preferable alga, as defined in the main text; black areas represent conditions under which selective feeding is not an optimal strategy even at the beginning of the day. Panel A is plotted under the assumption of a constant energy content (V) during the day, while panel B is plotted under the assumption of an increasing energy content function. The parameters used to produce the figures are: .</p

The diel feeding pattern disappears when the fish feed on algae grown protected from grazing.

<p>Average feeding rate (±SE) of <i>A. nigrofuscus</i> and <i>Z. xanthurum</i> on <b>A</b> non-manipulated and <b>B</b> protected plates measured 0.5–1.5 hr (AM, open bars) and 6–7 hrs (PM, full bars) after sunrise. Note the order of magnitude difference in scale between A and B, indicating the much higher bite rate on the protected plates.</p

Peptidoglycan maturation controls outer membrane protein assembly

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments(1–5). The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the β-barrel assembly machine(6,7) (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown(8). Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa(9), associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design