Establishment of a Vegetation Cover on Tundra Kimberlite Mine Tailings: 1. A Greenhouse Study

High erosion potential of dewatered kimberlite mine tailings after diamond extraction has prompted research at the Ekati Diamond Mine in the Canadian subarctic heath tundra ecosystem. Coarse texture, no organic component, lack of available macronutrients, and a serpentine chemistry are the principal limitations of these kimberlite tailings to plant colonization. Structure‐improving (peat moss, lake sediment, sewage sludge, Agri‐Boost, and composted papermill sludge) and nutrient‐providing (fertilizer, calcium carbonate, gypsum, and rock phosphate) amendments were tested in the greenhouse to ameliorate these limitations, thereby facilitating the field establishment of a permanent vegetation cover, which would stabilize the surface materials and promote natural colonization by the surrounding tundra vegetation. Seven native grass species (Arctagrostis latifolia, Calamagrostis canadensis, Poa glauca, Poa alpina, Deschampsia beringensis, Deschampsia caespitosa, and Festuca rubra) were used to measure amendment success. With the addition of structure‐improving and nutrient‐providing amendments, plant growth on kimberlite tailings was significantly enhanced. Tailings properties, including cation exchange capacity, percentage of organic carbon, and macronutrient availability, were also improved by amendment addition.     

Microbial Community Structure and Carbon-Utilization Diversity in a Mine Tailings Revegetation Study

Restoration of metals‐contaminated environments requires a functional microbial community for successful plant community establishment, soil development, and biogeochemical cycling. Our research measured microbial community structure and carbon‐utilization diversity in treatment plots from a mine waste revegetation project near Butte, Montana. Treatments included two controls (raw tailings) either (1) with or (2) without tilling, (3) shallow‐tilled lime addition, (4) deep‐tilled lime addition, (5) lime slurry injection, (6) topsoil addition, and (7) an undisturbed area near the tailings. Microbial community structural differences were assayed by plate counts of heterotrophic bacteria, actinomycetes, fungi, and bacterial endospores, and quantification of arbuscular mycorrhizae colonization. Metabolic diversity differences were assessed by carbon‐utilization profiles generated with Biolog microtiter plates. Heterotrophic bacteria counts were significantly higher in the limed and topsoil treatment plots than the control plots, and the actinomycete and fungal counts increased in the tilled control plot as well. Endospore counts were significantly higher in the topsoil addition and the undisturbed plots than the other treatment plots. Carbon‐utilization activity was very low in untreated plots, intermediate in lime‐treated plots, and very high in topsoil and undisturbed plots. Arbuscular mycorrhizae (AM) colonization levels of two grass species showed low levels of colonization on control, shallow‐limed, and lime slurry‐injected plots, and high levels on the deep‐limed and topsoil‐addition plots. Plant and soil system components increased across the treatment plots, but individual components responded differently to changing environmental conditions.     

Establishment of Restoration Trajectories for Upland Tundra Communities on Diamond Mine Wastes in the Canadian Arctic

Mining in the arctic amplifies restoration challenges due to inherent environmental conditions by removing soil, vegetation, and the propagule bank, adding coarse textured wastes with low water holding capacity and nutrients, and introducing salt and metal contamination. Short‐term reclamation focuses on rebuilding soil and providing rapid native plant cover for erosion control, supporting longer term reestablishment of ecological processes for sustainable tundra communities that provide essential wildlife habitat. This study evaluated methods to restore soil and plant communities 5 years after implementation of treatments at a diamond mine in the Canadian arctic. Five substrates including mine waste materials (processed kimberlite, glacial till, gravel, and mixes), four amendments (inorganic fertilizer, salvaged soil, sewage sludge, and water treatment sludge), five native species seed mixes and natural recovery were investigated. Soil and plant response were assessed annually. Soil chemistry was ameliorated with time. Chromium, cobalt, and nickel concentrations in processed kimberlite remained high and potentially toxic to plants. Adding fine textured materials such as glacial till to mine wastes improved nutrient and water retention, which in turn enhanced revegetation. Sewage and inorganic fertilizer increased available nitrogen and phosphorus, plant density and cover. Soil amendment increased species richness. Seeding was essential to establish a vegetation cover. After 5 years, seed mix composition and diversity had no effect on plant community development; soil and plant community properties among treatments changed considerably, providing evidence that restoration in the arctic is dynamic yet slow and success cannot be determined in the short term.     

The Use of Seedbed Modifications and Wood Chips to Accelerate Restoration of Well Pad Sites in Western Colorado,U.S.A.

Semiarid ecosystems of Western North America are experiencing a boom in natural gas development. However, these systems are slow to recover from the disturbances created. The purpose of this study was to develop improved restoration techniques on natural gas well pads in Western Colorado. This study examined effects and interactions of seedbed modifications, soil amendments, seed mixtures, and seeding methods. The experiment was conducted in pinyon‐juniper and semidesert shrub plant communities on five natural gas well pads beginning in 2006. Soil and plant cover data were collected to assess the effectiveness of 16 different treatment combinations. After two growing seasons, we found that patches of soil salinity (>4 dS/m) reduced plant cover to less than 20% on 55 of our 240 experimental plots. These patches of salinity, such as where reserve pits were buried, may need to be treated to completely restore cover on the total gas pad area, although causes of salinity patches needs further investigation. After removing the 55 saline plots from our data analyses, we found that wood chips (WC) as a soil amendment increased organic matter content and reduced non‐native species. Rough seedbed modifications increased the establishment of native species, especially during years of below average precipitation. Island broadcasting resulted in an increase of noxious plant cover during the second growing season. From these findings we recommend that disturbed well pads in a similar environment be restored by seeding native species on sites that are amended with WC and physically modified to create a roughened seedbed.     

Vegetation Response to Lime and Manure Compost Amendments on Acid Lead/Zinc Mine Tailings: A Greenhouse Study

Land disturbed by mining in China is a serious problem and lead/zinc (Pb/Zn) mine tailings constitute the majority of the metal mine tailings produced in Guangdaong Province, China. A greenhouse study was therefore conducted to evaluate the effects of lime (40, 80, 120, and 160 t/ha) and manure compost (50 and 100 t/ha) amendment on the revegetation of the Pb/Zn mine tailings using Cynodon dactylon (Bermuda grass) and Agropyron elongatum (tall wheatgrass). The results showed that a combination of lime and manure compost amendment together with deionized water leachating was able to increase pH, reduce electrical conductivity and diethylenetraminepentaacetic acid (DTPA)‐extractable concentrations of Zn and Pb in tailings. Using 80 t/ha lime amendment with the supplement of fertilizer or manure compost was able to effectively improve germination of both C. dactylon and A. elongatum. The highest dry weight yields were obtained in tailings receiving 80 t lime/ha and 100 t manure compost/ha for both plant species. Plant tissue analysis showed that lime amendment at 120–160 t/ha reduced Zn accumulation in both shoot and root of C. dactylon. However, this trend was not observed for Pb.     

Direct Effects of Soil Amendments on Field Emergence and Growth of the Invasive Annual Grass Bromus tectorum L. and the Native Perennial Grass Hilaria jamesii (Torr.) Benth

Bromus tectorum L. is a non-native, annual grass that has invaded western North America. In SE Utah, B. tectorum generally occurs in grasslands dominated by the native perennial grass, Hilaria jamesii (Torr.) Benth. and rarely where the natives Stipa hymenoides Roem. and Schult. and S. comata Trin. & Rupr. are dominant. This patchy invasion is likely due to differences in soil chemistry. Previous laboratory experiments investigated using soil amendments that would allow B. tectorum to germinate but would reduce B. tectorum emergence without affecting H. jamesii. For this study we selected the most successful treatments (CaCl₂, MgCl₂, NaCl and zeolite) from a previous laboratory study and applied them in the field in two different years at B. tectorum-dominated field sites. All amendments except the lowest level of CaCl₂ and zeolite negatively affected B. tectorum emergence and/or biomass. No amendments negatively affected the biomass of H. jamesii but NaCl reduced emergence. Amendment effectiveness depended on year of application and the length of time since application. The medium concentration of zeolite had the strongest negative effect on B. tectorum with little effect on H. jamesii. We conducted a laboratory experiment to determine why zeolite was effective and found it released large amounts of Na⁺, adsorbed Ca²⁺, and increased Zn²⁺, Fe²⁺, Mn²⁺, Cu²⁺, exchangeable Mg²⁺, exchangeable K, and NH₄⁺ in the soil. Our results suggest several possible amendments to control B. tectorum. However, variability in effectiveness due to abiotic factors such as precipitation and soil type must be accounted for when establishing management plans.     

Establishment of woody species across 26 years of revegetation on a Queensland coal mine

The establishment success of woody plant species at 56 revegetation sites, four to 26 years old, across the Meandu open‐cut coal mine in south‐east Queensland was assessed. The revegetation process involved returning stockpiled topsoil, deep ripping and mechanical sowing of a mix of native seeds. Blakes Wattle (Acacia blakei) and less often Black Wattle (A. leiocalyx), both primarily derived from respread topsoil seed, dominate the vegetation canopy at 59% and 20% of revegetation sites, respectively. The additional sowing of seeds of many tree and shrub species within the sites has had limited success with most failing to persist or grow well. Revegetation management, for example selective thinning of acacias (Acacia spp.) saplings within the first 5 years is recommended to release the competition pressure on the poorly performing tree species. This will also allow opportunities for other less well represented shrub and herb species to persist. This study has shown that a range of tree and shrub species, including Eucalyptus spp., are performing poorly under the current revegetation regime, suggesting adjustments to revegetation species selection and/or methodologies are needed. The natural colonization of woody native species within the sites from nearby remnant vegetation is shown to be limited to only four species, and therefore is unlikely to significantly supplement the species diversity of the revegetation.     

A Germination and Establishment Field Trial of Themeda australis (Kangaroo Grass) for Mine Site Restoration in the Central Tablelands of New South Wales

The current study demonstrated that Themeda australis (R. Br.) Stapf (kangaroo grass), a major understory component of the original grassy Box woodlands in the Central Tablelands of New South Wales, was suitable for use in large‐scale mine rehabilitation. The results of the trial were applied in the mine rehabilitation program. Due to extensive clearing of the woodlands and the introduction of exotic flora and fauna for agriculture, only small remnants of the original flora remain. The final land use of the gold mine is a conservation area free from agricultural pressure. Local native species adapted to the soil conditions and variable climate are highly desirable for the control of soil erosion following mining. Germination and establishment of T. australis on oxidized overburden were examined over 53 weeks. Seed‐bearing mulch was used as both the seeding material and an organic additive to the overburden. The effects of five factors were investigated: time since soil preparation, position within small contour banks, location of contour on experimental slopes, additional water, and rate of seed/mulch application. Direct seeding using the lowest rate of mulch application resulted in the establishment of more T. australis seedlings on new contours than on 15‐month‐old contours. The provision of additional water increased germination and establishment in both old and new contours, but was not essential. Seedling densities were greatest in the middle positions of contour banks up to week seven, but were destroyed during a heavy storm. At the close of the experiment, seedling densities were greater in the top and bottom positions of contours. Although significant slope × rate and age × slope interactions occurred, SNK tests did not reveal any consistent interpretable results.     

Enhanced phytoextraction: I. Effect of EDTA and citric acid on heavy metal mobility in a calcareous soil

Phytoextraction, the use of plants to extract heavy metals from contaminated soils, could be an interesting alternative to conventional remediation technologies. However, calcareous soils with relatively high total metal contents are difficult to phytoremediate due to low soluble metal concentrations. Soil amendments such as ethylene diaminetetraacetate (EDTA) have been suggested to increase heavy metal bioavailability and uptake in aboveground plant parts. Strong persistence of EDTA and risks of leaching of potentially toxic metals and essential nutrients have led to research on easily biodegradable soil amendments such as citric acid. In our research, EDTA is regarded as a scientific benchmark with which degradable alternatives are compared for enhanced phytoextraction purposes. The effects of increasing doses of EDTA (0.1,1,10 mmol kg(-1) dry soil) and citric acid (0.01, 0.05, 0.25, 0.442, 0.5 mol kg(-1) dry soil) on bioavailable fractions of Cu, Zn, Cd, and Pb were assessed in one part of our study and results are presented in this article. The evolution of labile soil fractions of heavy metals over time was evaluated using water paste saturation extraction (approximately soluble fraction), extraction with 1 M NH4OAc at pH 7 (approximately exchangeable fraction), and extraction with 0.5 M NH4OAc + 05 M HOAc + 0.02 M EDTA at pH 4.65 (approximately potentially bioavailable fraction). Both citric acid and EDTA produced a rapid initial increase in labile heavy metal fractions. Metal mobilization remained constant in time for soils treated with EDTA, but a strong exponential decrease of labile metal fractions was noted for soils treated with citric acid. The half life of heavy metal mobilization by citric acid varied between 1.5 and 5.7 d. In the following article, the effect of heavy metal mobilization on uptake by Helianthus annuus will be presented.     

Effects of N on Plant Response to Heat-wave: A Field Study with Prairie Vegetation

More intense, more frequent, and longer heat-waves are expected in the future due to global warming, which could have dramatic ecological impacts. Increasing nitrogen (N) availability and its dynamics will likely impact plant responses to heat stress and carbon (C) sequestration in terrestrial ecosystems. This field study examined the effects of N availability on plant response to heat-stress (HS) treatment in naturally-occurring vegetation. HS (5 d at ambient or 40.5 ℃) and N treatments (±N) were applied to 16 1 m2 plots in restored prairie vegetation dominated by Andropogon gerardii (warm-season C4 grass) and Solidago canadensis (warm-season C3 forb). Before, during, and after HS, air, canopy, and soil temperature were monitored; net CO2 assimilation (Pn), quantum yield of photosystem Ⅱ (φPsⅡ), stomatal conductance (gs), and leaf water potential (Ψw) of the dominant species and soil respiration (Rsolf) of each plot were measured daily during HS. One week after HS, plots were harvested, and C% and N% were determined for rhizosphere and bulk soil, and above-ground tissue (green/senescent leaf, stem, and flower). Photosynthetic N-use efficiency (PNUE) and N resorption rate (NRR) were calculated. HS decreased Pn, gs, Ψw, and PNUE for both species, and N treatment generally increased these variables (±HS), but often slowed their poat-HS recovery. Aboveground biomass tended to decrease with HS in both species (and for green leaf mass in S. canadensis), but decrease with N for ,4. gerardii and increase with N for S. canadensis. For A. gerardii, HS tended to decrease N% in green tissues with N, whereas in S. canadensis, HS increased N% in green leaves.Added N decreased NRR for A. gerardii and HS increased NRR for S. canadensis. These results suggest that heat waves,though transient, could have significant effects on plants, communities, and ecosystem N cycling, and N can influence the effect of heat waves.     

Early trajectories of forest understory development on reclamation sites: influence of forest floor placement and a cover crop

We tested whether direct placement of forest floor material (FFM: litter, fibric, humus layers and surface mineral horizons) and sowing of a cover crop (Melilotus officinalis) could facilitate the establishment of native forest understory species at a reclaimed coal mine in Alberta, Canada. FFM was salvaged at two depths (15 and 40 cm) from a recently harvested native aspen forest and immediately placed at the same depths on the reclamation site. Total richness (approximately 61 species in 96 subplots) was similar in each of 3 years post‐placement; total richness for all 3 years combined was 87 including 34 typical boreal forest understory species plus 30 other natives. The deeper treatment reduced cover of all species, native and non‐native species in year 1. In year 3, the deeper treatment still had lower cover of non‐native species but had higher cover of forest understory species in years 2 and 3. The deeper treatment also resulted in lower species richness per plot, but only in year 1. In year 2 (when the biennial clover was at its tall stage), the cover crop treatment was associated with lower cover of non‐native species but did not affect the cover of native forest understory species. Direct placement of FFM can help facilitate establishment of a diverse native boreal forest understory in a reclaimed landscape. Although richness and cover may be initially higher with shallower salvage and placement, deeper salvage may ultimately be better for encouraging establishment of native forest understory species.     

Primary Productivity and Water Balance of Grassland Vegetation on Three Soils in a Continuous CO2 Gradient: Initial Results from the Lysimeter CO2 Gradient Experiment

Field studies of atmospheric CO₂ effects on ecosystems usually include few levels of CO₂ and a single soil type, making it difficult to ascertain the shape of responses to increasing CO₂ or to generalize across soil types. The Lysimeter CO₂ Gradient (LYCOG) chambers were constructed to maintain a linear gradient of atmospheric CO₂ (~250 to 500 μl l⁻¹) on grassland vegetation established on intact soil monoliths from three soil series. The chambers maintained a linear daytime CO₂ gradient from 263 μl l⁻¹ at the subambient end of the gradient to 502 μl l⁻¹ at the superambient end, as well as a linear nighttime CO₂ gradient. Temperature variation within the chambers affected aboveground biomass and evapotranspiration, but the effects of temperature were small compared to the expected effects of CO₂. Aboveground biomass on Austin soils was 40% less than on Bastrop and Houston soils. Biomass differences between soils resulted from variation in biomass of Sorghastrum nutans, Bouteloua curtipendula, Schizachyrium scoparium (C₄ grasses), and Solidago canadensis (C₃ forb), suggesting the CO₂ sensitivity of these species may differ among soils. Evapotranspiration did not differ among the soils, but the CO₂ sensitivity of leaf-level photosynthesis and water use efficiency in S. canadensis was greater on Houston and Bastrop than on Austin soils, whereas the CO₂ sensitivity of soil CO₂ efflux was greater on Bastrop soils than on Austin or Houston soils. The effects of soil type on CO₂ sensitivity may be smaller for some processes that are tightly coupled to microclimate. LYCOG is useful for discerning the effects of soil type on the CO₂ sensitivity of ecosystem function in grasslands. Author Contributions: PF conceived study, analyzed data, and wrote the paper. AK, AP analyzed data. DH, VJ, RJ, HJ, and WP conceived study, and conducted research.     

Drivers of aboveground wood production in a lowland tropical forest of West Africa: teasing apart the roles of tree density,tree diversity,soil phosphorus,and historical logging

Tropical forests currently play a key role in regulating the terrestrial carbon cycle and abating climate change by storing carbon in wood. However, there remains considerable uncertainty as to whether tropical forests will continue to act as carbon sinks in the face of increased pressure from expanding human activities. Consequently, understanding what drives productivity in tropical forests is critical. We used permanent forest plot data from the Gola Rainforest National Park (Sierra Leone) – one of the largest tracts of intact tropical moist forest in West Africa – to explore how (1) stand basal area and tree diversity, (2) past disturbance associated with past logging, and (3) underlying soil nutrient gradients interact to determine rates of aboveground wood production (AWP). We started by statistically modeling the diameter growth of individual trees and used these models to estimate AWP for 142 permanent forest plots. We then used structural equation modeling to explore the direct and indirect pathways which shape rates of AWP. Across the plot network, stand basal area emerged as the strongest determinant of AWP, with densely packed stands exhibiting the fastest rates of AWP. In addition to stand packing density, both tree diversity and soil phosphorus content were also positively related to productivity. By contrast, historical logging activities negatively impacted AWP through the removal of large trees, which contributed disproportionately to productivity. Understanding what determines variation in wood production across tropical forest landscapes requires accounting for multiple interacting drivers – with stand structure, tree diversity, and soil nutrients all playing a key role. Importantly, our results also indicate that logging activities can have a long‐lasting impact on a forest's ability to sequester and store carbon, emphasizing the importance of safeguarding old‐growth tropical forests.     

The Influence of Soil Properties on the Mobility of Metals Following a Single Application of Polluted Sewage Sludge to Seventy Agricultural Topsoils: A Laboratory Column Study

The main aim of this study was to examine the influence of soil properties on the leaching of Cd, Cr, Cu, Ni, Pb, and Zn following the application of polluted sewage sludge to contrasting topsoils. Seventy agricultural soil samples from different parts of Spain were amended with a single dose of sewage sludge (equivalent to 50 t dry weight ha^?1) and a column study was performed under controlled conditions. After two, four, and six months of incubation, 283 ml of distilled water (equivalent to a rainfall event of 25 l m^?2) was applied. The leachates were then collected and analyzed for metals. For all of the soils considered, the pH was the most important parameter for the control of mobility metals (except for Cu, determined by the sand and soil organic carbon and only to a lesser extent by the soil pH r² = 0.604, p < 0.001) and was negatively related to all of the studied metals. For Pb and Zn, soil pH was the single soil property explaining their mobility (r² = 0.411, p < 0.001 for Pb; r² = 0.713, p < 0.001 for Zn) while for Cd, Cr and Ni, EC, sand and silt also appeared in the statistical models (r² = 0.753, p < 0.001 for Cd; r² = 0.366, p < 0.001 for Cr; r² = 0.784, p < 0.001 for Ni). In the basic soils, soil texture was the most important soil property controlling the mobility of metals (except for that of Pb, which it only weakly predicted). For the acidic-neutral soils, the soil pH was the most important soil property controlling metal mobility (except for that of Cr, which was mainly determined by the pseudo-total Cr content).     

Biodiversity and community structure of temperate butterfly species within a gradient of human disturbance: An analysis based on the concept of generalist vs. Specialist strategies

We monitored nine butterfly communities with varying degrees of human disturbance by conducting a census twice a month during 1980 by the line transect method in and around Tsukuba City, central Japan. We analyzed the biodiversity and community structures using the generalist/specialist concept. The site (community) order based on decreasing human disturbance was positively correlated with butterfly species diversity (H′), species richness (the total number of species), and the number of specialist species in a community, but not with the number of generalist species. The number of generalist species was rather constant, irrespective of the degree of human disturbance. Thus, both the butterfly species diversity and species richness were more dependent on the specialists than the generalists. Our analyses also showed that the generalist species were distributed widely over the communities, and they maintained high population densities, resulting in high rank status in abundance in a community, with more spatial variation in density per species. Specialist species showed the opposite trends. These results demonstrate that the generalist/specialist concept is a powerful tool applicable to analyse the biodiversity and structure of natural communities.     

间作对旱地CO2和N2O排放影响的研究进展

农田温室气体排放是近年来科学界的研究热点,采用合适的种植模式是减少农田温室气体排放的有效途径之一.本文综述了作物间作对旱地土壤CO₂和N₂O排放的影响及机理.合理间作能够提高土壤有机碳(SOC)含量、促进不同作物秸秆向SOC转化、降低SOC矿化速率,从而减少CO₂排放.禾本科与豆科作物间作能够在维持作物产量的情况下,减少化学氮肥投入、土壤有效氮残留及还田秸秆产生的无机氮,降低N₂O排放.间作作物的互作、田间小气候环境的改善也是影响土壤温室气体排放的重要因素.今后,要增加土壤温室气体监测时长并对影响因子进行综合、全面的分析,尤其是从分子水平探究间作模式下土壤微生物对温室气体产生过程的作用机理,为构建环境友好型农业模式提供科学依据.     

Strategy to design peptide inhibitors: structure of a complex of proteinase K with a designed octapeptide inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2 at 2.5 A resolution.

The crystal structure of a complex formed by the interaction between proteinase K and a designed octapeptide amide, N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2, has been determined at 2.5 A resolution and refined to an R-factor of 16.7% for 7,430 reflections in the resolution range of 8.0-2.50 A. The inhibitor forms a stable complex through a series of hydrogen bonds and hydrophobic interactions with the protein atoms and water molecules. The inhibitor is hydrolyzed between Phe4I and DAla5I (I indicates the inhibitor). The two fragments are separated by a distance of 3.2 A between the carbonyl carbon of Phe4I and the main-chain nitrogen of DAla5I. The N-terminal tetrapeptide occupies subsites S1-S5 (S5 for acetyl group), whereas the C-terminal part fits into S1'-S5' region (S5' for amide group). It is the first time that such an extended electron density for a designed synthetic peptide inhibitor has been observed in the prime region of an enzyme of the subtilisin family. In fact, the inhibitor fills the recognition site completely. There is only a slight rearrangement of the protein residues to accommodate the inhibitor. Superposition of the present octapeptide inhibitor on the hexapeptide inhibitor studied previously shows an overall homology of the two inhibitors, although the individual atoms are displaced significantly. It suggests the existence of a recognition site with flexible dimensions. Kinetic studies indicate an inhibition rate of 100% by this specifically designed peptide inhibitor.     

Ultrahigh resolution structure of a class A beta-lactamase: on the mechanism and specificity of the extended-spectrum SHV-2 enzyme

Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins. The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes. SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1. Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution. The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166. Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated. These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases. Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme. The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1. This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime.