Front Cover Image,Volume 116, Number 11, November 2019

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome ( www.ibiodesign.net/gBIG ).     

PHYSIOLOGICAL SNAPSHOTS REFLECT ECOLOGICAL PERFORMANCE OF THE SEA PALM,POSTELSIA PALMAEFORMIS (PHAEOPHYCAEA) ACROSS INTERTIDAL ELEVATION AND EXPOSURE GRADIENTS1

Postelsia palmaeformis Ruprecht is an intertidal kelp found only on very wave‐exposed rocky shores of the northeast Pacific. In areas dominated by mussels, Postelsia depends on wave‐induced disturbances to complete its life‐history cycle. Postelsia also recruits where mussels are absent, but not at less wave‐exposed shores. Thus, physical conditions related to wave exposure limit its horizontal distribution. It is not clear what limits the vertical distribution of Postelsia. We investigated factors contributing to Postelsia's limited distribution using transplant experiments, demographic monitoring, and field fluorometry to evaluate growth and performance across gradients of tidal elevation and wave exposure. Survivorship and growth were sharply reduced at upper and wave‐protected edges relative to mid‐level, wave‐exposed sporophytes. Reproductive output was reduced at upper and lower levels, and growth but not survivorship was lower at the lower level. Effects were independent of population of origin and were a manifestation of the environment. Maximum electron transport rates (ETR_m), light saturation parameters (E_k), and maximum quantum yields (ΔF/F_m) provided insight into physiological dynamics; all were lowest at the high edge, but increased when desiccation stress was alleviated by a mock sea‐spray treatment. The ETR_m and E_k values of low sporophytes were not as high as the values for mid‐sporophytes, despite higher or equivalent nitrogen content, chl a, and absorptance, suggesting a trade‐off between light‐capturing and carbon‐fixation capacity. Physiological limitations at upper and lower levels and deleterious desiccation effects at wave‐protected sites prevent establishment, thus constraining Postelsia to a mid‐zone, wave‐exposed distribution. Physical conditions related to wave exposure may limit the horizontal distribution of Postelsia because this kelp is also found in areas where mussels are lacking but not on less wave‐exposed shores.     

SINGLE‐CELL PULSE AMPLITUDE MODULATION FLUORESCENCE MEASUREMENTS OF THE GIANT DIATOM ETHMODISCUS (BACILLARIOPHYCEAE)1

Diurnal changes in effective yield (ΔF:F_m′), rapid light curves (RLCs), and induction/dark recovery time series were measured on individual cells of the giant diatom Ethmodiscus Castracane using active fluorescence (pulse amplitude modulation fluorometry). Unlike the co‐occurring diatom Hemiaulus and bulk phytoplankton, there was no observable diurnal down‐regulation of yield or relative electron transport rates in Ethmodiscus. Yields were constant at or near maximum values (0.7–0.8). Increases in ΔF:F_m′ during the initial actinic levels are consistent with dark nonphotochemical quenching mechanisms. Sustained actinic illumination (660 μmol photon·m^?2·s^?1) resulted in a ΔF:F_m′ of 0.2–0.3, but rapid recovery to near‐maximum values occurred in subsequent dark periods. Such recovery occurred even after exposure to full sunlight for 28 min, but not at 60 min. Thus, the lack of diurnal down‐regulation in Ethmodiscus is apparent, not real, and is an artifact of the time scale of sample extraction from net tows. These positively buoyant cells showed no evidence of routine photodamage, probably due to mixing and reduction in the average light exposure. The general patterns seen in RLCs from light‐and dark‐adapted higher plants are significantly different from those observed in Ethmodiscus. These results suggest that active fluorescence characteristics require careful examination to differentiate habitat‐ and taxon‐specific characteristics from light‐history effects. It is unclear whether the rapid recovery seen in Ethmodiscus is unique. The differences seen between Hemiaulus and Ethmodiscus from the same samples suggest that changes in community yield values measured in countertop systems could be the result of species replacement in addition to experimental or environmental perturbations.     

Consideration of the order Cryptonemiales and the families Nemastomataceae and Furcellariaceae (Gigartinales,Rhodophyta) in light of the morphology of Adelophyton corneum (J. Agardh) gen. et comb. nov. from southern Australia

A new red algal genus is described, based on the southern Australian Chaetangium corneum J. Agardh. It is reproductively unique in that while the auxiliary cell is intercalary in an adventitious filament, a defining character of the order Cryptonemiales, the vegetative structure, carpogonial branches, connecting filaments and gonimoblast development seem strongly allied to lower families of the Gigartinales. Although its predominantly thallus-inward gonimoblast development is characteristic of the gigartinalian family Furcellariaceae, it is suggested that the new alga should be placed in the Nemastomataceae and that this family exhibits features which, in an ancient algal stock, could theoretically have given rise to the lower cryptonemialian and higher gigartinalian lines. The possible relationships between the Nemastomataceae, Furcellariaceae and Solieriaceae are discussed, and some seemingly primitive features of the Nemastomataceae are enumcrated.     

油田含聚合物污水微生物处理初步研究

对油田日益增多的并且对环境造成潜在危害的而现有污水处理工艺难于处理的含聚合物污水进行微生物处理初步研究。结果表明,从油田污水污泥中分离得到的七株聚丙烯酰胺(PAM)降解菌在合适的营养条件下协同作用,对大港油田的含PAM的污水具有较好的处理效果。经过3d的处理,微生物菌群将补充了磷和氮的污水的COD降低了87.7%;经过18d的处理,微生物菌群将补充了磷的污水的COD由13499mg.L-1降低为283 mg.L-1,降低幅度达到了97.9%。因而这7株PAM降解菌对含PAM污水处理具有很好的应用前景。     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

Effect of sediment type on microphytobenthos vertical distribution: Modelling the productive biomass and improving ground truth measurements

Intertidal flats are frequently colonised by microphytobenthos (MPB) assemblages that form transient biofilms at the sediment surface which are responsible for large fractions of estuarine primary production. The large spatio-temporal variability in MPB biomass distribution in concert with the fact that tidal flats can cover many km² makes the use of remote sensing particularly useful in assessing MPB distribution. Water content, sediment type and MPB vertical migration are variables that affect the relationship between ground truth measurements and remote sensing of benthic chlorophyll. The effect of chlorophyll depth distribution (top 2 mm) on the relationship between benthic chlorophyll and several remote sensing indices (NDVI, PI, R562/R647, derivative indices and PAM fluorescence) was investigated over a 2 year sampling period at 6 sites (Tagus estuary, Portugal). Additionally, the effect of the dark adaptation time required to measure the minimum fluorescence parameter (F₀) was also tested. Sediment type strongly affected MPB depth distribution with muddy sites showing a strong negative exponential decay in chlorophyll with distance from the surface while sandy sites had a homogenous distribution over the same scale (2 mm). Chlorophyll content (mass per unit mass, μg g^− 1) in the top 2 mm was better correlated with remote sensing indices than concentration (mass per unit volume, mg m^− 3), both for NDVI (0.72 vs. 0.45) and for PAM fluorescence (0.70 vs. 0.55). Separating the data by transect increased the correlation values in all situations. A fitted model of chlorophyll depth distribution showed that the effect of asymmetrical chlorophyll depth distribution was stronger on the correlations between chlorophyll concentration and NDVI than on chlorophyll content and NDVI (0.46-2 mm vs. 0.74-125 μm, muddy site) the same was valid for fluorescence (0.66-2 mm vs. 0.92-125 μm, muddy site). Dark adapting the samples for more than 5 min did not result in any significant difference in the relationship between F₀ and chlorophyll a. The residuals from the regression of chlorophyll content on NDVI were positively correlated (0.7) with the mass per unit of mass of sediment < 63 μm and negatively (− 0.6) with chlorophyll concentration, this indicates that if no correction is performed to account for chlorophyll depth distribution both units will be strongly affected by the mass of < 63 μm particles. The results demonstrate that although expressing chlorophyll a as concentration is generally a better option for ground truth measurements care should be taken to account for chlorophyll depth distribution since strong asymmetries within the sampling depth can introduce large errors.     

Fe deficiency induces phosphorylation and translocation of Lhcb1 in barley thylakoid membranes

HvLhcb1 a major light-harvesting chlorophyll a/b-binding protein in barley, is a critical player in sustainable growth under Fe deficiency. Here, we demonstrate that Fe deficiency induces phosphorylation of HvLhcb1 proteins leading to their migration from grana stacks to stroma thylakoid membranes. HvLhcb1 remained phosphorylated even in the dark and apparently independently of state transition, which represents a mechanism for short-term acclimation. Our data suggest that the constitutive phosphorylation-triggered translocation of HvLhcb1 under Fe deficiency contributes to optimization of the excitation balance between photosystem II and photosystem I, the latter of which is a main target of Fe deficiency.