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SUMMARY Bacterial cell wall synthesis is the target for some of our most powerful antibiotics and has thus been the subject of intense research focus for more than 50 years. Surprisingly, we still lack a fundamental understanding of how bacteria build, maintain and expand their cell wall. Due to technical limitations, directly testing hypotheses about the coordination and biochemistry of cell wall synthesis enzymes or architecture has been challenging, and interpretation of data has therefore often relied on circumstantial evidence and implicit assumptions. A number of recent papers have exploited new technologies, like single molecule tracking and real-time, high resolution temporal mapping of cell wall synthesis processes, to address fundamental questions of bacterial cell wall biogenesis. The results have challenged established dogmas and it is therefore timely to integrate new data and old observations into a new model of cell wall biogenesis in rod-shaped bacteria.

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2015): Evaluating listening and speaking skills in a mobile game-based learning environment with situational contexts, Computer Assisted Language Learning, Game-based learning activities that facilitate students' listening and speaking skills were designed in this study. To participate in learning activities, students in the control group used traditional methods, while students in the experimental group used a mobile system. In our study, we looked into the feasibility of mobile game-based learning activities. One experiment was carried out and the results revealed that the experimental-group students significantly outperformed the control-group students on the verbal post-test. However, the performance of the two groups was equal on the listening post-test. Two variables (the average score for the interactive jigsaw game and the number of designed cards) were found to be the most important factors for influencing students' performance in the verbal post-test. Furthermore, most students had positive perceptions toward learning activities that are supported by a mobile system. These results suggest that game-based learning activities can significantly improve students' speaking skills if driven by a mobile system. Furthermore, these results suggest that learning activities with a mobile system foster students to (1) practice speaking English as a foreign language (EFL) more frequently as well as to reflect on their speech; (2) create meaningful sentences and speak with greater accuracy and confidence; and (3) practice speaking EFL in an authentic context.

Bacteria surround themselves with peptidoglycan, an adaptable enclosure that contributes to cell shape and stability. Peptidoglycan assembly relies on penicillin-binding proteins (PBPs) acting in concert with SEDS-family transglycosylases RodA and FtsW, which support cell elongation and division respectively. In Bacillus subtilis, cells lacking all four PBPs with transglycosylase activity (aPBPs) are viable. Here, we show that the alternative sigma factor σI is essential in the absence of aPBPs. Defects in aPBP-dependent wall synthesis are compensated by σI-dependent upregulation of an MreB homolog, MreBH, which localizes the LytE autolysin to the RodA-containing elongasome complex. Suppressor analysis reveals that cells unable to activate this σI stress response acquire gain-of-function mutations in the essential histidine kinase WalK, which also elevates expression of sigI, mreBH and lytE. These results reveal compensatory mechanisms that balance the directional peptidoglycan synthesis arising from the elongasome complex with the more diffusive action of aPBPs.

The integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. In Bacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C 55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimized clustered regularly interspaced short palindromic repeat (CRISPR) system with catalytically inactive ("dead") CRISPR-associated protein 9 (dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate that B. subtilis requires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the M -dependent cell envelope stress response, including bcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of the B. subtilis UPP-Pase enzymes, and provide further evidence linking the M regulon to cell envelope homeostasis pathways. IMPORTANCEThe emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

Heptaprenyl diphosphate (C35-PP) is an isoprenoid intermediate in the synthesis of both menaquinone and the sesquarterpenoids. We demonstrate that inactivation of ytpB, encoding a C35-PP utilizing enzyme required for sesquarterpenoid synthesis, leads to an increased sensitivity to bacitracin, an antibiotic that binds undecaprenyl pyrophosphate (C55-PP), a key intermediate in cell wall synthesis. Genetic studies indicate that bacitracin sensitivity is due to accumulation of C35-PP, rather than the absence of sesquarterpenoids. Sensitivity is accentuated in a ytpB menA double mutant, lacking both known C35-PP consuming enzymes, and in a ytpB strain overexpressing the HepST enzyme that synthesizes C35-PP. Conversely, sensitivity in the ytpB background is suppressed by mutation of hepST or by supplementation with 1,4-dihydroxy-2-naphthoate, a co-substrate with C35-PP for MenA. Bacitracin sensitivity results from impairment of the BceAB and BcrC resistance mechanisms by C35-PP: in a bceAB bcrC double mutant disruption of ytpB no longer increases bacitracin sensitivity. These results suggest that C35-PP inhibits both BcrC (a C55-PP phosphatase) and BceAB (an ABC transporter that confers bacitracin resistance). These findings lead to a model in which BceAB protects against bacitracin by transfer of the target, C55-PP, rather than the antibiotic across the membrane.

The surface erosion area in the Yangtze River basin increased from 364×10 3 km 2 in the 1950s to 707×10 3 km 2 in 2001 due to a great increase in population. Based on the regression relationship between surface erosion area and population, the surface erosion area was predicted to be about 280×103 km 2 at the beginning of the 20 th century. The sediment yield, which increased by about 30% during the first six decades of the 20 th century, was closely related to the surface erosion area in this river basin. The Yangtze annual suspended sediment flux into the estuary was about 395×106 t a -1 at the beginning of the century, and this gradually increased to an average of 509×10 6 t a -1 in the 1960s. The increase in the suspended sediment flux into the estuary was accelerated in the 1950s and the 1960s due to the rapid increase in population and land use immediately after the Second World War and the Liberation War. After the riverine suspended sediment flux reached its maximum in the 1960s, it decreased to <206×10 6 t a -1 in 2003. Construction of dams was found to be the principal cause for this decreasing trend because, during the same period, (a) the riverine water discharge did not show a decreasing trend, (b) water diversion was not influential and (c) sedimentation in lakes and canals of the middle and lower reaches did not increase. The total storage capacity of reservoirs has increased dramatically over the past half century. The amount of sediment trapped in reservoirs has increased to more than half a billion t a -1 . As a result, the suspended sediment flux into the estuary dramatically decreased, even though the sediment yield from many areas of the basin increased in recent decades. Human activities gradually increased the suspended sediment flux into the estuary before the 1960s and then rapidly decreased it. The last century was a period when the Yangtze suspended sediment flux into the estuary was dramatically affected by human activities.