Diel Vertical Dynamics of Gelatinous Zooplankton (Cnidaria, Ctenophora and Thaliacea) in a Subtropical Stratified Ecosystem (South Brazilian Bight).

The diel vertical dynamics of gelatinous zooplankton in physically stratified conditions over the 100-m isobath (~110 km offshore) in the South Brazilian Bight (26°45'S; 47°33'W) and the relationship to hydrography and food availability were analyzed by sampling every six hours over two consecutive days. Zooplankton samples were taken in three depth strata, following the vertical structure of the water column, with cold waters between 17 and 13.1°C, influenced by the South Atlantic Central Water (SACW) in the lower layer (>70 m); warm (>20°C) Tropical Water in the upper 40 m; and an intermediate thermocline with a deep chlorophyll-a maximum layer (0.3-0.6 mg m-3). Two distinct general patterns were observed, emphasizing the role of (i) physical and (ii) biological processes: (i) a strong influence of the vertical stratification, with most zooplankton absent or little abundant in the lower layer. The influence of the cold SACW on the bottom layer apparently restricted the vertical occupation of most species, which typically inhabit epipelagic warm waters. Even among migratory species, only a few (Aglaura hemistoma, Abylopsis tetragona eudoxids, Beroe sp., Thalia democratica, Salpa fusiformis) crossed the thermocline and reached the bottom layer. (ii) A general tendency of partial migrations, with variable intensity depending on the different species and developmental stages; populations tended to be more widely distributed through the water column during daylight, and to become more aggregated in the upper layer during the night, which can be explained based on the idea of the "hunger-satiation hypothesis", maximizing feeding and minimizing the chances of being predated.

The mangotoxin biosynthetic operon (mbo) is specifically distributed within Pseudomonas syringae genomospecies 1 and was acquired only once during evolution.

Mangotoxin production was first described in Pseudomonas syringae pv. syringae strains. A phenotypic characterization of 94 P. syringae strains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair specific for the mbo operon to examine its distribution within the P. syringae complex. These primers amplified a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor the mbo operon, whereas 35 non-mangotoxin-producing strains did not yield any amplification. This, together with the analysis of draft genomes, allowed the identification of the mbo operon in five pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of the mbo genes in the P. syringae complex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing the mbo operon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. The mbo operon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that the mbo operon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within the P. syringae complex.

Pyk2 regulates H+-ATPase-mediated proton secretion in the outer medullary collecting duct via an ERK1/2 signaling pathway.

Acid-secreting intercalated cells respond to changes in systemic pH through regulation of apical H(+) transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pH(o)). Pyk2 is a nonreceptor tyrosine kinase activated by autophosphorylation at Tyr402 by cell-specific stimuli, including decreased pH, and is involved in the regulation of MAPK signaling pathways and transporter activity. We examined whether the Pyk2 and MAPK signaling pathway mediates the response of transport proteins to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 small interfering RNA to knockdown Pyk2 expression levels, the Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP 1) to inhibit Pyk2 phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. The pH-sensitive fluorescent probe 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was used to assay H(+) transporter activity. The activity of H(+) transporters was measured as the rate of intracellular pH (pH(i)) recovery after an NH(4)Cl prepulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media [extracellular pH (pH(o)) 6.7] increased the phosphorylation of Pyk2, ERK1/2, and p38. Reduction in pH(i) induced by an NH(4)Cl prepulse also increased the phosphorylation of Pyk2, ERK1/2, and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H(+)-ATPase and H(+),K(+)-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 phosphorylation as well as H(+)-ATPase-mediated recovery in mOMCD1 cells. In addition, ERK1/2 inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H(+)-ATPase-mediated pH(i) recovery but not phosphorylation of p38. We conclude that Pyk2 and ERK1/2 are required for increasing H(+)-ATPase, but not H(+),K(+)-ATPase, activity at decreased pH(i) in mOMCD1 cells.

A-kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle.

A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules that can compartmentalize cAMP signaling transduced by ß2-adrenergic receptors (ß(2)ARs); such compartmentalization ensures speed and fidelity of cAMP signaling and effects on cell function. This study aimed to assess the role of AKAPs in regulating global and compartmentalized ß(2)AR signaling in human airway smooth muscle (ASM). Transcriptome and proteomic analyses were used to characterize AKAP expression in ASM. Stable expression or injection of peptides AKAP-IS or Ht31 was used to disrupt AKAP-PKA interactions, and global and compartmentalized cAMP accumulation stimulated by ß-agonist was assessed by radioimmunoassay and membrane-delineated flow through cyclic nucleotide-gated channels, respectively. ASM expresses multiple AKAP family members, with gravin and ezrin among the most readily detected. AKAP-PKA disruption had minimal effects on whole-cell cAMP accumulation stimulated by ß-agonist (EC(50) and B(max)) concentrations, but significantly increased the duration of plasma membrane-delineated cAMP (τ=251±51 s for scrambled peptide control vs. 399±79 s for Ht31). Direct PKA inhibition eliminated decay of membrane-delineated cAMP levels. AKAPs coordinate compartmentalized cAMP signaling in ASM cells by regulating multiple elements of ß(2)AR-mediated cAMP accumulation, thereby representing a novel target for manipulating ß(2)AR signaling and function in ASM.

pH-dependent regulation of the α-subunit of H+-K+-ATPase (HKα2).

The H(+)-K(+)-ATPase α-subunit (HKα(2)) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα(2) plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing G(s)-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα(2) expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO(2). PKA activity and HKα(2) protein were monitored by immunoblot analysis, and HKα(2) mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα(2) mRNA; however, PKA activity and HKα(2) protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα(2) expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα(2) protein by increasing PKA activity without altering HKα(2) mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα(2) and acid-base balance.

Contribution of mangotoxin to the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae.

Mangotoxin is an antimetabolite toxin that inhibits ornithine acetyl transferase, a key enzyme in the biosynthetic pathway of ornithine and arginine and recently reported in strains of Pseudomonas syringae pv. syringae (Pss) isolated from mango. Since symptoms on mango tissues are very difficult to reproduce, in this study the role of mangotoxin in Pss virulence was addressed by analyzing the in planta growth and development of disease symptoms on tomato leaflets. Inoculation experiments were carried out following several procedures using the wild-type strain Pss UMAF0158, two Tn5-mutant derivative strains defective in mangotoxin production, and their complemented derivative strains in which mangotoxin production is restored. The ability of the mangotoxin-defective mutants to grow in planta was similar, and their epiphytic survival on the tomato leaf surface identical to the wild-type and complemented strains. However, both the disease index data of incidence and the severity of necrotic symptoms indicated that mangotoxin-defective mutants were less virulent, indicating that mangotoxin is a virulence factor. Furthermore, competition experiments showed that the survival values of the wild-type strain were slightly but significantly higher than those of the mangotoxin-defective mutants, suggesting that mangotoxin production would improve the epiphytic fitness of Pss.

Contribution of mangotoxin to the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae

Mangotoxin is an antimetabolite toxin that inhibits ornithine acetyl transferase, a key enzyme in the biosynthetic pathway of ornithine and arginine and recently reported in strains of Pseudomonas syringae pv. syringae (Pss) isolated from mango. Since symptoms on mango tissues are very difficult to reproduce, in this study the role of mangotoxin in Pss virulence was addressed by analyzing the in planta growth and development of disease symptoms on tomato leaflets. Inoculation experiments were carried out following several procedures using the wild-type strain Pss UMAF0158, two Tn5-mutant derivative strains defective in mangotoxin production, and their complemented derivative strains in which mangotoxin production is restored. The ability of the mangotoxin-defective mutants to grow in planta was similar, and their epiphytic survival on the tomato leaf surface identical to the wild-type and complemented strains. However, both the disease index data of incidence and the severity of necrotic symptoms indicated that mangotoxin-defective mutants were less virulent, indicating that mangotoxin is a virulence factor. Furthermore, competition experiments showed that the survival values of the wild-type strain were slightly but significantly higher than those of the mangotoxin-defective mutants, suggesting that mangotoxin production would improve the epiphytic fitness of Pss (AU)

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Comparative histochemical analyses of oxidative burst and cell wall reinforcement in compatible and incompatible melon-powdery mildew (Podosphaera fusca) interactions.

The spatial-temporal expression patterns of oxidative burst and cell wall reinforcement were analyzed in leaves of resistant and susceptible melon (Cucumis melo L.) cultivars in response to Podosphaera fusca (Fr.) Braun & Shishkoff, the main causal agent of powdery mildew in cucurbits. Extensive development of powdery mildew mycelia and a progressive increase in haustorial count were recorded in the susceptible cultivar after 4d, while in the resistant cultivar powdery mildew failed to grow and small brownish and necrotic leaf areas were frequently observed. Rapid generation of the reactive oxygen intermediates hydrogen peroxide and superoxide radicals 4h after pathogen challenge, but before the fungal haustoria formation, stood upstream in the cascade of events induced during these interactions. This oxidative burst was followed by the accumulation of strengthening polymers of callose and lignin at the cell wall of attacked resistant plant cells. Interestingly, the transcriptional levels of phenylalanine ammonia-lyase (PAL), an important enzyme for phenylpropanoid metabolism, did not significantly change throughout the experiments. Although these physiological changes were observed in both cultivars, their faster kinetics and amplitude in the resistant line compared to the susceptible cultivar governed the differential visual response of these cultivars against P. fusca. These findings, along with data obtained in previous studies, have provided the bases for an integrated model in which the spatial-temporal response patterns of these resistance mechanisms have been arranged, which may ultimately lead to successful protection of melon plants against P. fusca.

Molecular regulation and physiology of the H+,K+ -ATPases in kidney.

Two H(+), K(+)-adenosine triphosphatase (ATPase) proteins participate in K(+) absorption and H(+) secretion in the renal medulla. Both the gastric (HKalpha(1)) and colonic (HKalpha(2)) H(+),K(+)-ATPases have been localized and characterized by a number of techniques, and are known to be highly regulated in response to acid-base and electrolyte disturbances. Both ATPases are dimers of composition alpha/beta that localize to the apical membrane and both interact with the tetraspanin protein CD63. Although CD63 interacts with the carboxy-terminus of the alpha-subunit of the colonic H(+),K(+)-ATPase, it interacts with the beta-subunit of the gastric H(+),K(+)-ATPase. Pharmacologically, both ATPases are distinct; for example, the gastric H(+),K(+)-ATPase is inhibited by Sch-28080, but the colonic H(+),K(+)-ATPase is inhibited by ouabain (a classic inhibitor of the Na(+)-pump) and is completely insensitive to Sch-28080. The alpha-subunit of the colonic H(+),K(+)-ATPase is the only subunit of the X(+),K(+)-ATPase superfamily that has 3 different splice variants that emerge by deletion or elongation of the amino-terminus. The messenger RNA and protein of one of these splice variants (HKalpha(2C)) is specifically up-regulated in newborn rats and becomes undetectable in adult rats. Therefore, HKalpha(2), in addition to its role in potassium and acid-base homeostasis, appears to play a significant role in early growth and development. Finally, because chronic hypokalemia appears to be the most potent stimulus for upregulation of HKalpha(2), we propose that the HKalpha(2) participates importantly in the maintenance of chronic metabolic alkalosis.

Phosphorylation of S955 at the protein kinase A consensus promotes maturation of the alpha subunit of the colonic H+,K+ -ATPase.

All the alpha subunits of the Na+,K+ -ATPases and H+,K+ -ATPases have a protein kinase A (PKA) consensus sequence near or in the ninth transmembrane domain. The role of this domain in influencing alpha subunit synthesis/degradation, plasma membrane localization, and 86Rb+ uptake has not been established for the alpha subunit of the colonic H+,K+ -ATPase. This study examined the effect of mutating S955 (within the PKA consensus site of the alpha subunit of the colonic H+,K+ -ATPase [HKalpha2]) to alanine (S955/A) or aspartic acid (S955/D) on alpha subunit expression and function. The results demonstrate that a negatively charged amino acid at position 955 of HKalpha2 promotes higher expression levels of both whole-cell and plasma membrane-localized HKalpha2. Moreover, inhibition of PKA reduced expression of wild-type HKalpha2 and associated 86Rb+ uptake. Last, the activity of the HKalpha2 S955/A was rescued by treatment with 4-phenylbutyric acid, a compound that was shown previously to restore function to the cystic fibrosis transmembrane conductance regulator.

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to decrease [corrected] plasma membrane localization and 86Rb+ uptake.

The carboxy terminus (CT) of the colonic H(+)-K(+)-ATPase is required for stable assembly with the beta-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKalpha(2), we selected 84 amino acids in the CT of the alpha-subunit of mouse colonic H(+)-K(+)-ATPase (CT-HKalpha(2)) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HKalpha(2) with CD63, recombinant CT-HKalpha(2) and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HKalpha(2) protein (but not CT-HKalpha(1)) coprecipitated with CD63, confirming stable assembly of HKalpha(2) with CD63. In HEK-293 transfected with HKalpha(2) plus beta(1)-Na(+)-K(+)-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKalpha(2)/NKbeta(1) and (86)Rb(+) uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKalpha(2)-NKbeta(1) complex in the cell membrane.

A carboxy-terminus motif of HKalpha2 is necessary for assembly and function.

BACKGROUND: The present experiments were designed to study the importance of the carboxy-terminus of HKalpha2, for both function and integrity of assembly with beta1-Na+,K+-ATPase. METHODS: For this purpose, stop codons were created, by polymerase chain reaction (PCR), at different positions in the carboxy-terminus of HKalpha2. Subsequently, chimeras between HKalpha2 and the carboxy-terminus of alpha1-Na+,K+-ATPase or with the carboxy-terminus of the gastric H+,K+-ATPase were created. Human embryonic kidney HEK-293 cells were used as expression systems for functional studies using 86Rb+ uptake and alpha/beta assembly using specific antibodies. RESULTS: The results demonstrate that the entire carboxy-terminus of HKalpha2 is required for optimal protection of the alpha/beta complex from degradation and for functionality as evidenced by 86Rb+ uptake. The results also demonstrate that there was flexibility in the sequence of the carboxy-terminus. The last two tyrosines (Y1035Y1036) of HKalpha2 could be mutated to alanines and the carboxy-terminus of HKalpha2 could be replaced by the carboxy-terminus of alpha1-Na+,K+-ATPase while preserving transport activity. CONCLUSION: The entire carboxy-terminus of HKalpha2 is required for stable assembly with beta1-Na+,K+-ATPase and functionality.

The effect of beta-subunit assembly on function and localization of the colonic H+,K+-ATPase alpha-subunit.

BACKGROUND: Previous experiments from our laboratory have demonstrated that HKalpha(2) coimmunoprecipitated with beta(1)-Na(+),K(+)-ATPase. Although HKalpha(2) is expressed abundantly in the apical membrane of distal colon, the demonstration that beta(1) localizes to this same membrane in distal colon has not been demonstrated previously. METHODS: Immunolocalization was performed in distal colon using a polyclonal antibody against HKalpha(2) and a monoclonal antibody against beta(1). RESULTS: The results demonstrate that HKalpha(2) localizes to the apical membrane. Two pools of beta(1)-Na(+),K(+)-ATPase were detected. The first localized to the apical membrane. The second pool was detected in the basolateral membrane when distal colon sections were deglycosylated with glycosidase F. Therefore, our results demonstrate that beta(1) localizes to the apical membrane with HKalpha(2), and supports the view that beta(1) is the physiologic beta-subunit for HKalpha(2). We tested, therefore, the efficiency of the two beta-subunits expressed in distal colon (beta(1) and beta(3)) to support the activity of HKalpha(2). Human embryonic kidney HEK-293 cells were transiently cotransfected with HKalpha(2) plus beta(1) or HKalpha(2) plus beta(3). Subsequently, (86)Rb(+)-uptake and plasma membrane localization were evaluated. The results demonstrate that both HKalpha(2)/beta(1) and HKalpha(2)/beta(3) support (86)Rb(+)-uptake. However, (86)Rb(+)-uptake measured in the cells cotransfected with HKalpha(2) plus beta(1) exceeded that measured in cells expressing HKalpha(2)/beta(3). Fluorescence microscopy using enhanced green fluorescent protein cloned at the amino-terminus of HKalpha(2) demonstrated protein migration to the plasma membrane in cells cotransfected with EGFP-HKalpha(2) plus beta(1). In contrast, in cells cotransfected with EGFP-HKalpha(2) plus beta(3), the vast majority of the protein remained confined to intracellular compartments. The significantly higher (86)Rb(+)-uptake corresponded to additional localization of HKalpha(2) to the plasma membrane when coexpressed with beta(1) compared to beta(3). CONCLUSION: Taken together, these and previous results from our laboratory indicate that beta(1)-Na(+),K(+)-ATPase is likely to represent the most physiologic and efficient subunit for HKalpha(2) assembly in distal colon.

The carboxy terminus of the colonic H(+), K(+)-ATPase alpha-subunit is required for stable beta subunit assembly and function.

BACKGROUND: The present experiments were designed to study the importance of the carboxy-terminus of colonic H(+), K(+)-ATPase alpha-subunit (HKalpha(2)), for both function as well as integrity of assembly with beta1-Na(+), K(+)-ATPase. METHODS: For this purpose, a mutation of 84 amino acids in the carboxy-terminus was created (DeltaHKalpha(2)) and HEK-293 cells were used as expression systems for functional studies using (86)Rb(+)-uptake, coimmunoprecipitation using specific antibodies and fluorescence microscopy using green fluorescent protein. RESULTS: The results demonstrate that comparable levels of expression of HKalpha(2) and DeltaHKalpha(2) mRNA were observed when cells were cotransfected with beta1 subunit. However, the abundance of expression of full length HKalpha(2) protein exceeded that of the truncated protein DeltaHKalpha(2). Ouabain-sensitive (86)Rb(+)-uptake was present only in cells cotransfected with HKalpha(2)/beta(1), indicating that the mutation was incapable of sustaining functionality. Coimmunoprecipitation experiments demonstrated that HKalpha(2) protein was immunoprecipitated more abundantly than DeltaHKalpha(2) when coexpressed with beta1. The use of sucrose gradients and green fluorescence protein immunofluorescence demonstrated that while the DeltaHKalpha(2)/beta(1) complex was confined to the endoplasmic reticulum, the HKalpha(2)/beta(1) complex translocated to the plasma membrane. CONCLUSION: Taken together, our results are consistent with the view that the carboxy-terminus of HKalpha(2) facilitates the proper folding of the HKalpha(2)/beta(1) complex allowing translocation of the heterodimer to the plasma membrane where potassium uptake occurs. Otherwise, the alpha/beta complex is destined for degradation.

The gamma-Na+,K+-ATPase subunit assembles selectively with alpha1/beta1-Na+,K+-ATPase but not with the colonic H+,K+-ATPase.

BACKGROUND: The ubiquitous Na+-pump (Na+,K+-ATPase) assembles as a heterodimer of composition alpha/beta in some nephron segments, while in other segments it may exist as a heterotrimer of composition alpha/beta/gamma. The gamma-subunit has been reported to increase the affinity of the Na+-pump for adenosine 5'-triphosphate (ATP), and decrease affinity for both Na+ and K+. The alpha-subunit of the colonic H+,K+-ATPase (cHK) shares 75% sequence similarity with alpha1-Na+,K+-ATPase (alpha1) and assembles with beta1-Na+,K+-ATPase (beta1) in distal colon and renal medulla. Differences in pharmacological properties have been ascribed to when heterologously expressed function has been compared to function in vitro. The purpose of this study was to determine if cHK might associate with the gamma-subunit of the Na+,K+-ATPase (gamma) as a possible explanation for these variations in function. METHODS: An antibody specific for the gamma was used in coimmunoprecipitation experiments to determine if the gamma assembles stably in vitro with cHK and beta1 in rat renal medulla or distal colon. RESULTS: Our results demonstrate that the gamma-subunit assembles specifically with the Na+-pump, but not with cHK. Furthermore, the gamma-subunit assembly was specific for rat kidney and was not observed in distal colon. CONCLUSION: Since the gamma-subunit did not assemble with the cHK/beta1 complex, gamma-subunit assembly cannot explain those variations in ex vivo and in vitro pharmacologic properties ascribed to cHK.

Copper Resistance in Pseudomonas syringae Strains Isolated from Mango Is Encoded Mainly by Plasmids.

ABSTRACT Bacterial apical necrosis of mango, elicited by Pseudomonas syringae pv. syringae, limits fruit production in southern Spain and Portugal. Examination of a collection of P. syringae pv. syringae isolates for copper resistance showed that 59% were resistant to cupric sulfate. The survey of a mango orchard revealed an increase in frequencies of copper-resistant bacteria after repeated treatments with Bordeaux mixture. These data suggest that selection of copper-resistant strains could be a major reason for control failures following management with copper bactericides. Most copper-resistant isolates harbored plasmids, although the majority of them contained a 62-kb plasmid that also was present in copper-sensitive strains. The 62-kb plasmids were differentiated by restriction enzyme analysis and hybridization to copABCD DNA. The most frequently found copper-resistant plasmid type (62.1) was transferable by conjugation. Southern blot hybridizations showed that genetic determinants partially homologous to copABCD were present in all the copper-resistant strains examined, and usually were associated with plasmids; these determinants were not detected in copper-sensitive strains. The selective pressure exerted by copper bactericide sprays on the diversity of copper resistance determinants in bacterial populations of mango is discussed.