The Challenges of Characterizing the Zooplankton Community Response Following Simulated Spills of Diluted Bitumen into Boreal Lake Limnocorrals.

We attempted to characterize zooplankton community response following spills of the unconventional crude oil, diluted bitumen (dilbit), into 10-m diameter, ~ 100 m3, ~ 1.5-m deep boreal lake limnocorrals, including two controls and seven dilbit treatments ranging from 1.5 to 180 L (1:100,000 to 1:1,000 v/v, dilbit:water). Community composition and abundances were monitored weekly to bi-weekly over three months. Total zooplankton biomass and abundance seemingly collapsed in all limnocorrals, regardless of treatment, though some rotifer species persisted. As a result, it was not possible to determine the impacts of dilbit. We theorize several potential non-oil-related reasons for the sudden community collapse - including elevated zinc levels, fish grazing pressures, and sampling biases - and provide guidance for future work using in-lake enclosures.

Surface oil is the primary driver of macroinvertebrate impacts following spills of diluted bitumen in freshwater.

The response of freshwater invertebrates following accidental releases of oil is not well understood. This knowledge gap is more substantial for unconventional oils such as diluted bitumen (dilbit). We evaluated the effects of dilbit on insect emergence and benthic invertebrates by conducting experimental spills in limnocorrals (10-m diameter; ~100-m3) deployed in a boreal lake at the IISD-Experimental Lakes Area, Canada. The study included seven dilbit treatments (spill volumes ranged from 1.5 L [1:66,000, oil:water, v/v] to 180 L [1:590, oil:water, v/v]), two controls, and additional lake reference sites, monitored for 11 weeks. Invertebrate emergence declined at the community level following oil addition in a significantly volume-dependent manner, and by 93-100 % over the 11 weeks following the spill in the highest treatment. Dilbit altered community structure of benthic invertebrates, but not abundance. One-year post-spill and following oil removal using traditional skimming and absorption techniques, benthic richness and abundance were greater among all treatments than the previous year. These results indicate that recovery in community composition is possible following oil removal from a lake ecosystem. Research is needed concerning the mechanisms by which surface oil directly affect adult invertebrates, whether through limiting oviposition, limiting emergence, or both. The response of benthic communities to sediment tar mats is also warranted.

Changes in net ecosystem productivity of boreal black spruce stands in response to changes in temperature at diurnal and seasonal time scales.

Net ecosystem productivity (NEP) of boreal coniferous forests is believed to rise with climate warming, thereby offsetting some of the rise in atmospheric CO(2) concentration (C(a)) by which warming is caused. However, the response of conifer NEP to warming may vary seasonally, with rises in spring and declines in summer. To gain more insight into this response, we compared changes in CO(2) exchange measured by eddy covariance and simulated by the ecosystem process model ecosys under rising mean annual air temperatures (T(a)) during 2004-2006 at black spruce stands in Saskatchewan, Manitoba and Quebec. Hourly net CO(2) uptake was found to rise with warming at T(a) < 15 degrees C and to decline with warming at T(a) > 20 degrees C. As mean annual T(a) rose from 2004 to 2006, increases in net CO(2) uptake with warming at lower T(a) were greater than declines with warming at higher T(a) so that annual gross primary productivity and hence NEP increased. Increases in net CO(2) uptake measured at lower T(a) were explained in the model by earlier recovery of photosynthetic capacity in spring, and by increases in carboxylation activity, using parameters for the Arrhenius temperature functions of key carboxylation processes derived from independent experiments. Declines in net CO(2) uptake measured at higher T(a) were explained in the model by sharp declines in mid-afternoon canopy stomatal conductance (g(c)) under higher vapor pressure deficits (D). These declines were modeled from a hydraulic constraint to water uptake imposed by low axial conductivity of conifer roots and boles that forced declines in canopy water potential (psi(c)), and hence in g(c) under higher D when equilibrating water uptake with transpiration. In a model sensitivity study, the contrasting responses of net CO(2) uptake to specified rises in T(a) caused annual NEP of black spruce in the model to rise with increases in T(a) of up to 6 degrees C, but to decline with further increases at mid-continental sites with lower precipitation. However, these contrasting responses to warming also indicate that rises in NEP with climate warming would depend on the seasonality (spring versus summer) as well as the magnitude of rises in T(a).

Changes in net ecosystem productivity with forest age following clearcutting of a coastal Douglas-fir forest: testing a mathematical model with eddy covariance measurements along a forest chronosequence.

We hypothesized that changes in net ecosystem productivity (NEP) during aging of coastal Douglas-fir (Pseudotsuga menziesii Mirb. Franco) stands could be explained by (1) changing nutrient uptake caused by different time scales for decomposition of fine, non-woody and coarse woody litter left after harvesting, (2) declines in canopy water status with lengthening of the water uptake pathway during bole and branch growth, and (3) increases in the ratio of autotrophic respiration (R (a)) to gross primary productivity (GPP) with phytomass accumulation. These hypotheses were implemented and tested in the mathematical model ecosys against eddy covariance (EC) measurements of forest CO(2) and energy exchange in a post-clearcut Douglas-fir chronosequence. Hypothesis 1 explained how (a) an initial rise in GPP observed during the first 3 years after clearcutting could be caused by nutrient mineralization from rapid decomposition of fine, non-woody litter with lower C:N ratios (assart effect), (b) a slower rise in GPP during the next 20 years could be caused by immobilization during later decomposition of coarse woody litter, and (c) a rapid rise in GPP between 20 and 40 years after clearcutting could be caused by nutrient mineralization with further decomposition of coarse woody litter and of its decomposition products. During periods (a) and (b), heterotrophic respiration (R (h)) from decomposition of fine and coarse litter greatly exceeded net primary productivity (NPP = GPP - R (a)) so that Douglas-fir stands were large sources of CO(2). During period (c), NPP exceeded R (h) so that these stands became large sinks for CO(2). Hypothesis 2 explained how declines in NPP during later growth in period (c) could be caused by lower hydraulic conductances in taller trees that would force lower canopy water potentials and hence greater sensitivity of stomatal conductances and CO(2) uptake to vapor pressure deficits. Enhanced sensitivity to vapor pressure deficits was also apparent in the EC measurements over the post-clearcut chronosequence. Hypothesis 3 did not contribute to the explanation of forest age effects on NEP.

Species-specific differences in positive and negative regulatory elements in the renin gene enhancer.

A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4. 1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences.

Expression of a renin/GFP transgene in mouse embryonic, extra-embryonic, and adult tissues.

A reporter construct was assembled with 4-kb of renin 5'-flanking sequence fused to humanized green fluorescent protein (GFP) cDNA. Transgenic mice carrying this construct were produced and assayed for GFP expression. In the adult, expression was detected in juxtaglomerular (JG) cells of the kidney and granular convoluted tubular cells of the submandibular gland. Furthermore, treatment of mice with captopril induced GFP expression in renal vascular smooth muscle cells. During embryogenesis, GFP expression was first detected at embryonic day E13 in the adrenal gland and Wolffian duct. Expression was also seen in the developing renal vasculature as early as E14 and remained detectable through birth. Renal GFP expression became restricted to JG cells in adults. Fetal adrenal and gonadal arteries also expressed GFP. In the placenta, GFP was observed in giant cell trophoblasts, consistent with reports of renin expression in chorionic cells of both humans and mice. We conclude that 4 kb of renin 5' flank is sufficient to direct multiple known renin expression patterns. Furthermore, the renin-GFP construct characterized here will provide a useful vital reporter for renin expression.

Climate and vegetation controls on boreal zone energy exchange.

The boreal forest, one of the world's larger biomes, is distinct from other biomes because it experiences a short growing season and extremely cold winter temperatures. Despite its size and impact on the earth's climate system, measurements of mass and energy exchange have been rare until the past five years. This paper overviews results of recent and comprehensive field studies conducted in Canada, Siberia and Scandinavia on energy exchanges between boreal forests and the atmosphere. How the boreal biosphere and atmosphere interact to affect the interception of solar energy and how solar energy is used to evaporate water and heat the air and soil is examined in detail. Specifically, we analyse the magnitudes, temporal and spatial patterns and controls of solar energy, moisture and sensible heat fluxes across the land-atmosphere interface. We interpret and synthesize field data with the aid of a soil-vegetation-atmosphere transfer model, which considers the coupling of the energy and carbon fluxes and nutrient status. Low precipitation and low temperatures limit growth of many boreal forests. These factors restrict photosynthetic capacity and lower root hydraulic conductivity and stomatal conductance of the inhabitant forests. In such circumstances, these factors interact to form a canopy that has a low leaf area index and exerts a significant resistance to evaporation. Conifer forests, growing on upland soils, for example, evaporate at rates between 25 and 75% of equilibrium evaporation and lose less than 2.5 mm day-1 of water. The open nature of many boreal conifer forest stands causes a disproportionate amount of energy exchange to occur at the soil surface. The climatic and physiological factors that yield relatively low rates of evaporation over conifer stands also cause high rates of sensible heat exchange and the diurnal development of deep planetary boundary layers. In contrast, evaporation from broad-leaved aspen stands and fen/wetlands approach equilibrium evaporation rates and lose up to 6 mm day-1 .

Low stomatal and internal conductance to CO2 versus Rubisco deactivation as determinants of the photosynthetic decline of ageing evergreen leaves.

A novel A-Ci curve (net CO2 assimilation rate of a leaf -An- as a function of its intercellular CO2 concentration -Ci) analysis method (Plant, Cell & Environment 27, 137-153, 2004) was used to estimate the CO2 transfer conductance (gi) and the maximal carboxylation (Vcmax) and electron transport (Jmax) potentials of ageing, non-senescing Pseudotsuga menziesii leaves in relation to their nitrogen (N) content and protein and pigment composition. Both gi and the stomatal conductance (gsc) of leaves were closely coupled to Vcmax, Jmax and An with all variables decreasing with increasing leaf age. Consequently, both Ci and Cc (chloroplastic CO2 concentration) remained largely conserved through successive growing seasons. The N content of leaves, as well as the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and other sodium dodecyl sulfate-soluble proteins, increased during the first three growing seasons, then stabilized or decreased only slightly afterwards. Thus, the age-related photosynthetic nitrogen use efficiency (PNUE) decline of leaves was not a consequence of decreased allocation of N towards Rubisco and other proteins involved in bioenergetics and light harvesting. Rather, loss of photosynthetic capacity was the result of the decreased activation state of Rubisco and proportional down-regulation of electron transport towards the photosynthetic carbon reduction (PCR) and photorespiratory (PCO) cycles in response to a reduction of CO2 supply to the chloroplasts' stroma. This study emphasizes the regulatory potential and homeostaticity of Cc- rather than photosynthetic metabolites or Ci- in relation to the commonly observed correlation between photosynthesis and gsc.

In vitro activities of posaconazole, fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically important molds and yeasts.

The in vitro activity of the novel triazole antifungal agent posaconazole (Noxafil; SCH 56592) was assessed in 45 laboratories against approximately 19,000 clinically important strains of yeasts and molds. The activity of posaconazole was compared with those of itraconazole, fluconazole, voriconazole, and amphotericin B against subsets of the isolates. Strains were tested utilizing Clinical and Laboratory Standards Institute broth microdilution methods using RPMI 1640 medium (except for amphotericin B, which was frequently tested in antibiotic medium 3). MICs were determined at the recommended endpoints and time intervals. Against all fungi in the database (22,850 MICs), the MIC(50) and MIC(90) values for posaconazole were 0.063 microg/ml and 1 mug/ml, respectively. MIC(90) values against all yeasts (18,351 MICs) and molds (4,499 MICs) were both 1 mug/ml. In comparative studies against subsets of the isolates, posaconazole was more active than, or within 1 dilution of, the comparator drugs itraconazole, fluconazole, voriconazole, and amphotericin B against approximately 7,000 isolates of Candida and Cryptococcus spp. Against all molds (1,702 MICs, including 1,423 MICs for Aspergillus isolates), posaconazole was more active than or equal to the comparator drugs in almost every category. Posaconazole was active against isolates of Candida and Aspergillus spp. that exhibit resistance to fluconazole, voriconazole, and amphotericin B and was much more active than the other triazoles against zygomycetes. Posaconazole exhibited potent antifungal activity against a wide variety of clinically important fungal pathogens and was frequently more active than other azoles and amphotericin B.

Predicting the onset of net carbon uptake by deciduous forests with soil temperature and climate data: a synthesis of FLUXNET data.

We tested the hypothesis that the date of the onset of net carbon uptake by temperate deciduous forest canopies corresponds with the time when the mean daily soil temperature equals the mean annual air temperature. The hypothesis was tested using over 30 site-years of data from 12 field sites where CO(2) exchange is being measured continuously with the eddy covariance method. The sites spanned the geographic range of Europe, North America and Asia and spanned a climate space of 16 degrees C in mean annual temperature. The tested phenology rule was robust and worked well over a 75 day range of the initiation of carbon uptake, starting as early as day 88 near Ione, California to as late as day 147 near Takayama, Japan. Overall, we observed that 64% of variance in the timing when net carbon uptake started was explained by the date when soil temperature matched the mean annual air temperature. We also observed a strong correlation between mean annual air temperature and the day that a deciduous forest starts to be a carbon sink. Consequently we are able to provide a simple phenological rule that can be implemented in regional carbon balance models and be assessed with soil and temperature outputs produced by climate and weather models.

Analysis of a Het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing.

Transposon-generated mutant N10 of Anabaena sp. strain PCC 7120 has a Het- phenotype (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation reproduced a Het- phenotype, but reconstructions with other insertions at the position of the transposon produced strains that form multiple contiguous heterocysts. Sequence analysis around the site of insertion of the transposon showed that the insertion lies within the 5' end of an 861-bp open reading frame (ORF) (hetN). The product of translation of hetN (HetN) shows extensive similarity to NAD(P)H-dependent oxidoreductases that are involved in biosyntheses of fatty acids, poly-beta-hydroxybutyrate, nod factor, and polyketides. A second, 1,518-bp ORF (hetM) that ends 556 bp 5' from the start of hetN appears to encode a protein that has at least two functional domains: its amino terminus is similar to an acyl carrier protein, while its central portion is similar to domains of proteins that perform reductive reactions. A third, 711-bp ORF (hetI) encoded on the opposite strand ends 42 bp away from the 3' end of hetN. The protein encoded by hetI, HetI, is similar to Sfp from Bacillus subtilis and EntD from Escherichia coli, proteins that are required for the biosynthesis or export of cyclic peptides. Clones from a lambda-EMBL3 library that contain the wild-type DNA for hetN do not complement the hetN::Tn5-1063 mutation in N10. The presence of hetN, as the only ORF, on a replicating plasmid suppresses heterocyst formation in wild-type cells, whereas the additional presence of hetI alleviates this effect.

Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena.

The spatially patterned differentiation of heterocysts in the filamentous cyanobacterium Anabaena requires a functional hetR gene. Transcriptional fusions to luxAB show that hetR is transcribed at a low level throughout the filament when Anabaena is grown with combined nitrogen, and that induction of the gene begins within 2 h following nitrogen deprivation. By 3.5 h, induction is localized to spaced foci. By 6 h, there is an overall induction of at least threefold in whole cultures, reflecting at least a 20-fold increase within spatially separated cells. The induction requires the presence of a functional hetR gene, indicating that hetR is autoregulatory. Full induction of a heterocyst structural gene, hepA, also requires a functional hetR locus.

High-resolution mapping of genetic loci of Anabaena PCC 7120 required for photosynthesis and nitrogen fixation.

A physical map of the Anabaena genome permitted the localization of its genes to chromosomal fragments generated by rarely cutting restriction endonucleases and separated by pulsed-field gel electrophoresis. We introduce a novel means of mapping more precisely to c. 20 kb by use of rare restriction sites within vectors bearing cloned sequences that undergo homologous recombination with the genome. We thereby localize and orient genes encoding principal photosynthetic pigments. The relative spacing of loci within a single restriction fragment was determined with even higher resolution, as illustrated for genes required for heterocyst development and nitrogen fixation that were marked with transposons. Small, newly visualized restriction fragments of the chromosome were also mapped.

Regulation of an osmoticum-responsive gene in Anabaena sp. strain PCC 7120.

Salt-induced genes in the cyanobacterium Anabaena sp. strain PCC 7120 were identified by use of a Tn5-based transposon bearing luxAB as a reporter. The genomic sequence adjacent to one site of insertion of the transposon was identical in part to the sequence of the lti2 gene, which was previously identified in a differential screen for cold-induced transcripts in Anabaena variabilis. The lti2-like gene was induced by sucrose and other osmotica and by low temperature, in addition to salt. Regulatory components necessary for the induction of this gene by osmotica were sought by a further round of transposon mutagenesis. One mutant that displayed reduced transcriptional activity of the lti2-like gene in response to exposure to osmotica had an insertion in an open reading frame, which was denoted orrA, whose predicted product showed sequence similarity to response regulators from two-component regulatory systems. The corresponding mutation was reconstructed and was shown, like the second-site transposon mutation, to result in reduced response to osmotic stress. Induction of the lux reporter gene by osmotica was restored by complementation with a genomic fragment containing the entire open reading frame for the presumptive response regulator, whereas a fragment containing a truncated copy of the open reading frame for the response regulator did not complement the mutation.

Cyclic mechanical distension regulates renin gene transcription in As4.1 cells.

The renin-producing and -secreting juxtaglomerular (JG) cells are thought to function as the baroreceptor of the kidney. The mechanism by which changes in pressure, or mechanical force, regulate renin at the molecular level has not been elucidated. The renin gene-expressing and -secreting clonal cell line As4.1 was derived from transgene-targeted oncogenesis in mice and was used as a cellular model for JG cells. As4.1 cells subjected to cyclic mechanical distension for a period of 24 h at various frequencies (0. 05 or 0.5 Hz) and magnitudes (12 or 24% elongation) were analyzed via Northern analysis for renin mRNA levels. Results indicate that renin gene expression is decreased by 50-85% and returns to basal levels after a 24-h recovery period. Renin gene expression was attenuated independently of elevated cell growth or changes in renin message decay, suggesting that renin gene transcription is directly modulated by mechanical distension. Transient transfection of As4.1 cells with renin 5' flanking sequence-luciferase reporter gene constructs confirmed the role of mechanical stimulation in regulating renin gene transcription. A 43% inhibition of luciferase activity, by stretch, was observed in cells transfected with a 4,000 base pair 5' flanking sequence to the renin proximal promoter. These results demonstrate for the first time that changes in mechanical force can result in the regulation of renin gene transcription and thus provide further insight into the baroreceptor properties of renin-expressing cells.

Endotoxin-induced renal inflammatory response. Oncostatin M as a major mediator of suppressed renin expression.

The systemic response to endotoxin is characterized by hypotension and severe reductions in blood pressure, leading to cardiovascular collapse that can accompany septicemia. The renin/angiotensin system would normally be expected to respond to hypotensive challenge; however, inflammation appears to modify this response. This study identifies a strong acute phase response of the kidney that is characterized by enhanced expression of serum amyloid A, haptoglobin and tissue inhibitor for metalloproteinase-1 and a reduced expression of renin. Equivalent regulatory effects were observed for the immortalized As4.1 kidney cell line that models certain features of juxtaglomerular cells. Oncostatin M, a known endotoxin-responsive proinflammatory cytokine, proved to be an effective inhibitor of renin gene expression. Suppression by oncostatin M involves activated STAT5 and requires an inhibitory element in the renin promoter that functions separately from cell type-specific enhancer elements. The renal acute phase reaction, unlike the liver acute phase reaction, is more strongly dependent on locally produced inflammatory factors.

An Abd-B class HOX.PBX recognition sequence is required for expression from the mouse Ren-1c gene.

Expression from the mouse Ren-1(c) gene in As4.1 cells is dependent on a proximal promoter element (PPE) located at approximately -60 and a 241-base pair enhancer region located at -2625 relative to the transcription start site. The PPE (TAATAAATCAA) is identical to a consensus HOX.PBX binding sequence. Further, PBX1b has been shown to be a component of a PPE-specific binding complex present in nuclear extracts from As4.1 cells. The binding affinities of different paralog HOX members to the PPE were examined in the absence or presence of PBX1b. HOXB6, -B7, and -C8 failed to bind the PPE alone but showed weak affinity in the presence of PBX1b. In contrast, HOXD10 and to a lesser degree HOXB9 bound the PPE with high affinities regardless of whether PBX1b was present. Abd-B HOX members, including HOXD10, -A10, -A9, -B9, and -C9, are expressed in As4.1 cells. The ability of HOX and PBX1b to form a ternary complex with PREP1 on the PPE is also demonstrated both in vivo and in vitro. Point mutations in either the HOX or PBX half-site of the PPE disrupted the formation of the HOX.PBX complex and dramatically decreased transcriptional activity of the Ren-1(c) gene demonstrating that both the HOX and PBX half-sites are critical for mouse renin gene expression. These results strongly implicate Abd-B class Hox genes and their cofactors as major determinants of the sites of renin expression.

Mutations in ribosomal protein L16 conferring reduced susceptibility to evernimicin (SCH27899): implications for mechanism of action.

A clinical isolate of Streptococcus pneumoniae (SP#5) that showed decreased susceptibility to evernimicin (MIC, 1.5 microgram/ml) was investigated. A 4,255-bp EcoRI fragment cloned from SP#5 was identified by its ability to transform evernimicin-susceptible S. pneumoniae R6 (MIC, 0.03 microgram/ml) such that the evernimicin MIC was 1.5 microgram/ml. Nucleotide sequence analysis of this fragment revealed that it contained portions of the S10-spc ribosomal protein operons. The nucleotide sequences of resistant and susceptible isolates were compared, and a point mutation (thymine to guanine) that causes an Ile52-Ser substitution in ribosomal protein L16 was identified. The role of this mutation in decreasing susceptibility to evernimicin was confirmed by direct transformation of the altered L16 gene. The presence of the L16 mutation in the resistant strain suggests that evernimicin is an inhibitor of protein synthesis. This was confirmed by inhibition studies using radiolabeled substrates, which showed that the addition of evernimicin at sub-MIC levels resulted in a rapid decrease in the incorporation of radiolabeled isoleucine in a susceptible isolate (SP#3) but was much less effective against SP#5. The incorporation of isoleucine showed a linear response to the dose level of evernimicin. The incorporation of other classes of labeled substrates was unaffected or much delayed, indicating that these were secondary effects.

Effects of mutations in ribosomal protein L16 on susceptibility and accumulation of evernimicin.

Chemical mutagenesis of Staphylococcus aureus RN450 generated two strains that displayed a stable reduction (30- to 60-fold) in susceptibility to evernimicin. Cell-free translation reactions demonstrated that the resistance determinant was located in the ribosomal fraction. Compared to ribosomes isolated from a wild-type strain, ribosomes from the mutant strains displayed an 8- to 10-fold reduction in affinity for [(14)C]evernimicin. In contrast, the mutants displayed no alteration in either binding affinity or in vitro susceptibility to erythromycin. Exponential cultures of the mutant strains accumulated significantly less [(14)C]evernimicin than the wild-type strain, suggesting that accumulation is dependent on the high affinity that evernimicin displays for its binding site. Sequencing rplP (encodes ribosomal protein L16) in the mutant strains revealed a single base change in each strain, which resulted in a substitution of either cysteine or histidine for arginine at residue 51. Introduction of a multicopy plasmid carrying wild-type rplP into the mutant strains restored sensitivity to evernimicin, confirming that the alterations in rplP were responsible for the change in susceptibility. Overexpression of the mutant alleles in S. aureus RN450 had no effect on susceptibility to evernimicin, demonstrating that susceptibility is dominant over resistance.