Unexpected Reactions of α,ß-Unsaturated Fatty Acids Provide Insight into the Mechanisms of CYP152 Peroxygenases.

CYP152 peroxygenases catalyze decarboxylation and hydroxylation of fatty acids using H2 O2 as cofactor. To understand the molecular basis for the chemo- and regioselectivity of these unique P450 enzymes, we analyze the activities of three CYP152 peroxygenases (OleTJE , P450SPα , P450BSß ) towards cis- and trans-dodecenoic acids as substrate probes. The unexpected 6S-hydroxylation of the trans-isomer and 4R-hydroxylation of the cis-isomer by OleTJE , and molecular docking results suggest that the unprecedented selectivity is due to OleTJE 's preference of C2-C3 cis-configuration. In addition to the common epoxide products, undecanal is the unexpected major product of P450SPα and P450BSß regardless of the cis/trans-configuration of substrates. The combined H218 O2 tracing experiments, MD simulations, and QM/MM calculations unravel an unusual mechanism for Compound I-mediated aldehyde formation in which the active site water derived from H2 O2 activation is involved in the generation of a four-membered ring lactone intermediate. These findings provide new insights into the unusual mechanisms of CYP152 peroxygenases.

How external perturbations affect the chemoselectivity of substrate activation by cytochrome P450 OleTJE.

Cytochrome P450 enzymes are versatile biocatalysts found in most forms of life. Generally, the cytochrome P450s react with dioxygen and hence are haem-based mono-oxygenases; however, in specific isozymes, H2O2 rather than O2 is used and these P450s act as peroxygenases. The P450 OleTJE is a peroxygenase that binds long to medium chain fatty acids and converts them to a range of products originating from Cα-hydroxylation, Cß-hydroxylation, Cα-Cß desaturation and decarboxylation of the substrate. There is still controversy regarding the details of the reaction mechanism of P450 OleTJE; how the products are formed and whether the product distributions can be influenced by external perturbations. To gain further insights into the structure and reactivity of P450 OleTJE, we set up a range of large active site model complexes as well as full enzymatic structures and did a combination of density functional theory studies and quantum mechanics/molecular mechanics calculations. In particular, the work focused on the mechanisms leading to these products under various reaction conditions. Thus, for a small cluster model, we find a highly selective Cα-hydroxylation pathway that is preferred over Cß-H hydrogen atom abstraction by at least 10 kcal mol-1. Introduction of polar residues to the model, such as an active site protonated histidine residue or through external electric field effects, lowers the Cß-H hydrogen atom abstraction barriers are lowered, while a full QM/MM model brings the Cα-H and Cß-H hydrogen atom abstraction barriers within 1 kcal mol-1. Our studies; therefore, implicate that environmental effects in the second-coordination sphere can direct and guide selectivities in enzymatic reaction mechanisms.

Production of Propene from n-Butanol: A Three-Step Cascade Utilizing the Cytochrome P450 Fatty Acid Decarboxylase OleTJE.

Propene is one of the most important starting materials in the chemical industry. Herein, we report an enzymatic cascade reaction for the biocatalytic production of propene starting from n-butanol, thus offering a biobased production from glucose. In order to create an efficient system, we faced the issue of an optimal cofactor supply for the fatty acid decarboxylase OleTJE , which is said to be driven by either NAD(P)H or H2 O2 . In the first system, we used an alcohol and aldehyde dehydrogenase coupled to OleTJE by the electron-transfer complex putidaredoxin reductase/putidaredoxin, allowing regeneration of the NAD+ cofactor. With the second system, we intended full oxidation of n-butanol to butyric acid, generating one equivalent of H2 O2 that can be used for the oxidative decarboxylation. As the optimal substrate is a long-chain fatty acid, we also tried to create an improved variant for the decarboxylation of butyric acid by using rational protein design. Within a mutational study with 57 designed mutants, we generated the mutant OleTV292I , which showed a 2.4-fold improvement in propene production in our H2 O2 -driven cascade system and reached total turnover numbers >1000.

The integrase of the Macrococcus caseolyticus resistance island mecD (McRImecD ) inserts DNA site-specifically into Staphylococcus and Bacillus chromosomes.

The methicillin resistance gene mecD has been recently identified on chromosomal islands in Macrococcus caseolyticus (McRImecD ). The 5' end of McRImecD carries an integrase (int) of the tyrosine recombinase family and two genes (intR and xis) encoding putative DNA-binding proteins. The islands are integrated site-specifically at the 3' end of the rpsI gene, a highly conserved locus in Gram-positive bacteria. Moreover, the rpsI gene of some Staphylococcus and Bacillus strains was found to be followed by a related integrase, raising the question of whether McRImecD could be transferred to these species. We used circular model elements carrying 5' end fragments of McRImecD -1 to demonstrate that the int enzyme and the attachment (att) site were sufficient to mediate site-specific DNA integration into the rpsI locus of Staphylococcus aureus, Staphylococcus pseudintermedius and Bacillus thuringiensis in vivo. Including xis in the model element stimulated both integrative and excisive recombination reactions and influenced the Int enzyme in att site selection. The intR gene functions as a negative regulator of int and xis. The int-xis genes of McRImecD -1 encode a site-specific recombination function that enables the acquisition of McRImecD in new hosts and the potential dissemination of broad-spectrum ß-lactam resistance across genus barriers.

Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleTJE.

The cytochrome P450 enzyme OleTJE from Jeotgalicoccus sp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of H2O2 as cofactor or using redox partner systems. This enzyme has attracted much attention due to its intriguing but unclear catalytic mechanism and potential application in biofuel production. Here, we investigated the functionality of a select group of residues (Arg245, Cys365, His85, and Ile170) in the active site of OleTJE through extensive mutagenesis analysis. The key roles of these residues for catalytic activity and reaction type selectivity were identified. In addition, a range of heterologous redox partners were found to be able to efficiently support the decarboxylation activity of OleTJE. The best combination turned out to be SeFdx-6 (ferredoxin) from Synechococcus elongatus PCC 7942 and CgFdR-2 (ferredoxin reductase) from Corynebacterium glutamicum ATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleTJE towards myristic acid were determined.

Production of alkenes and novel secondary products by P450 OleTJE using novel H2 O2 -generating fusion protein systems.

Jeotgalicoccus sp. 8456 OleTJE (CYP152L1) is a fatty acid decarboxylase cytochrome P450 that uses hydrogen peroxide (H2 O2 ) to catalyse production of terminal alkenes, which are industrially important chemicals with biofuel applications. We report enzyme fusion systems in which Streptomyces coelicolor alditol oxidase (AldO) is linked to OleTJE . AldO oxidizes polyols (including glycerol), generating H2 O2 as a coproduct and facilitating its use for efficient OleTJE -dependent fatty acid decarboxylation. AldO activity is regulatable by polyol substrate titration, enabling control over H2 O2 supply to minimize oxidative inactivation of OleTJE and prolong activity for increased alkene production. We also use these fusion systems to generate novel products from secondary turnover of 2-OH and 3-OH myristic acid primary products, expanding the catalytic repertoire of OleTJE .

Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE.

The Jeotgalicoccus sp. peroxygenase cytochrome P450 OleTJE (CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleTJE could enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences. In this manuscript, we have focused on OleTJE active site residues Phe79, His85, and Arg245 to interrogate their roles in substrate binding and catalytic activity. His85 is a potential proton donor to reactive iron-oxo species during substrate decarboxylation. The H85Q mutant substitutes a glutamine found in several peroxygenases that favor fatty acid hydroxylation. H85Q OleTJE still favors alkene production, suggesting alternative protonation mechanisms. However, the mutant undergoes only minor substrate binding-induced heme iron spin state shift toward high spin by comparison with WT OleTJE, indicating the key role of His85 in this process. Phe79 interacts with His85, and Phe79 mutants showed diminished affinity for shorter chain (C10-C16) fatty acids and weak substrate-induced high spin conversion. F79A OleTJE is least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg245 is crucial for binding the substrate carboxylate, and R245E/L mutations severely compromise activity and heme content, although alkene products are formed from some substrates, including stearic acid (C18:0). The results identify crucial roles for the active site amino acid trio in determining OleTJE catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation.

Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.

Catalytic strategy for carbon-carbon bond scission by the cytochrome P450 OleT.

OleT is a cytochrome P450 that catalyzes the hydrogen peroxide-dependent metabolism of Cn chain-length fatty acids to synthesize Cn-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe(4+)-oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C-H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe(4+)-OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon-carbon scission reaction catalyzed by OleT.

Expanding the substrate scope and reactivity of cytochrome P450 OleT.

The efficient hydrogen peroxide-dependent hydroxylation and epoxidation of hydrocarbons is catalysed by a P450 fatty acid decarboxylase (OleT) active-site variant. The introduction of an acidic functionality in the protein framework circumvents the necessity for a carboxylate that is typically provided by the substrate for efficient H2O2 heterolysis. Spectroscopic and turnover studies show that the mutation eliminates the binding and metabolism of prototypical fatty acid substrates, but permits the oxidation of a broad range of inert hydrocarbon substrates.

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. Objective : This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities. .

Enzyme(s) responsible for tellurite reducing activity in a moderately halophilic bacterium, Salinicoccus iranensis strain QW6.

Oxyanions of tellurium, like tellurate (TeO4 (2-)) and tellurite (TeO3 (2-)), are highly toxic for most microorganisms. There are a few reports on the bacterial tellurite resistance mechanism(s). Salinicoccus iranensis, a Gram-positive halophilic bacterium, shows high tellurite resistance and NADH-dependent tellurite reduction activity in vitro. Since little is known regarding TeO3 (2-) resistance mechanisms in halophilic microorganisms, here one of the enzymatic reduction activities presented in this microorganism is investigated. To enhance the enzymatic activity during purification, the effect of different parameters including time, inoculation, different pHs, different tellurite concentrations and different salts were optimized. We also examined the tellurite removal rates by diethyldithiocarbamate (DDTC) during optimization. In the culture medium the optimum conditions obtained showed that at 30 h, 2 % inoculum, pH 7.5, without tellurite and with 5 % NaCl (w/v) the highest enzyme activity and tellurite removal were observed. Results of the purification procedure done by hydroxyapatite batch-mode, ammonium sulfate precipitation, followed by phenyl-Sepharose and Sephadex G-100 column chromatography, showed that the enzyme consisted of three subunits with molecular masses of 135, 63 and 57 kDa. In addition to tellurite reduction activity, the enzyme was able to reduce nitrate too. Our study extends the knowledge regarding this process in halophilic microorganisms. Besides, this approach may suggest an application for the organism or the enzyme itself to be used for bioremediation of polluted areas with different contaminants due to its nitrate reductase activity.

Structure and biochemical properties of the alkene producing cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 bacterium.

The production of hydrocarbons in nature has been documented for only a limited set of organisms, with many of the molecular components underpinning these processes only recently identified. There is an obvious scope for application of these catalysts and engineered variants thereof in the future production of biofuels. Here we present biochemical characterization and crystal structures of a cytochrome P450 fatty acid peroxygenase: the terminal alkene forming OleTJE (CYP152L1) from Jeotgalicoccus sp. 8456. OleTJE is stabilized at high ionic strength, but aggregation and precipitation of OleTJE in low salt buffer can be turned to advantage for purification, because resolubilized OleTJE is fully active and extensively dissociated from lipids. OleTJE binds avidly to a range of long chain fatty acids, and structures of both ligand-free and arachidic acid-bound OleTJE reveal that the P450 active site is preformed for fatty acid binding. OleTJE heme iron has an unusually positive redox potential (-103 mV versus normal hydrogen electrode), which is not significantly affected by substrate binding, despite extensive conversion of the heme iron to a high spin ferric state. Terminal alkenes are produced from a range of saturated fatty acids (C12-C20), and stopped-flow spectroscopy indicates a rapid reaction between peroxide and fatty acid-bound OleTJE (167 s(-1) at 200 µm H2O2). Surprisingly, the active site is highly similar in structure to the related P450BSß, which catalyzes hydroxylation of fatty acids as opposed to decarboxylation. Our data provide new insights into structural and mechanistic properties of a robust P450 with potential industrial applications.

Studying and polishing the PDB's macromolecules.

Macromolecular crystal structures are among the best of scientific data, providing detailed insight into these complex and biologically important molecules with a relatively low level of error and subjectivity. However, there are two notable problems with getting the most information from them. The first is that the models are not perfect: there is still opportunity for improving them, and users need to evaluate whether the local reliability in a structure is up to answering their question of interest. The second is that protein and nucleic acid molecules are highly complex and individual, inherently handed and three-dimensional, and the cooperative and subtle interactions that govern their detailed structure and function are not intuitively evident. Thus there is a real need for graphical representations and descriptive classifications that enable molecular 3D literacy. We have spent our career working to understand these elegant molecules ourselves, and building tools to help us and others determine and understand them better. The Protein Data Bank (PDB) has of course been vital and central to this undertaking. Here we combine some history of our involvement as depositors, illustrators, evaluators, and end-users of PDB structures with commentary on how best to study and draw scientific inferences from them.

Comparative genome analysis of central nitrogen metabolism and its control by GlnR in the class Bacilli.

BACKGROUND: The assimilation of nitrogen in bacteria is achieved through only a few metabolic conversions between alpha-ketoglutarate, glutamate and glutamine. The enzymes that catalyze these conversions are glutamine synthetase, glutaminase, glutamate dehydrogenase and glutamine alpha-ketoglutarate aminotransferase. In low-GC Gram-positive bacteria the transcriptional control over the levels of the related enzymes is mediated by four regulators: GlnR, TnrA, GltC and CodY. We have analyzed the genomes of all species belonging to the taxonomic families Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae to determine the diversity in central nitrogen metabolism and reconstructed the regulation by GlnR. RESULTS: Although we observed a substantial difference in the extent of central nitrogen metabolism in the various species, the basic GlnR regulon was remarkably constant and appeared not affected by the presence or absence of the other three main regulators. We found a conserved regulatory association of GlnR with glutamine synthetase (glnRA operon), and the transport of ammonium (amtB-glnK) and glutamine/glutamate (i.e. via glnQHMP, glnPHQ, gltT, alsT). In addition less-conserved associations were found with, for instance, glutamate dehydrogenase in Streptococcaceae, purine catabolism and the reduction of nitrite in Bacillaceae, and aspartate/asparagine deamination in Lactobacillaceae. CONCLUSIONS: Our analyses imply GlnR-mediated regulation in constraining the import of ammonia/amino-containing compounds and the production of intracellular ammonia under conditions of high nitrogen availability. Such a role fits with the intrinsic need for tight control of ammonia levels to limit futile cycling.

Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus.

A putative fatty acid hydratase gene from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was a 68 kDa dimer with a molecular mass of 136 kDa. The enzymatic products formed from fatty acid substrates by the putative enzyme were isolated with high purity (>99%) by solvent fractional crystallization at low temperature. After the identification by GC-MS, the purified hydroxy fatty acids were used as standards to quantitatively determine specific activities and kinetic parameters for fatty acids as substrates. Among the fatty acids evaluated, specific activity and catalytic efficiency (k(cat)/K(m)) were highest for oleic acid, indicating that the putative fatty acid hydratase was an oleate hydratase. Hydration occurred only for cis-9-double and cis-12-double bonds of unsaturated fatty acids without any trans-configurations. The maximum activity for oleate hydration was observed at pH 6.5 and 25 °C with 2% (v/v) ethanol and 0.2 mM FAD. Without FAD, all catalytic activity was abolished. Thus, the oleate hydratase is an FAD-dependent enzyme. The residues G29, G31, S34, E50, and E56, which are conserved in the FAD-binding motif of fatty acid hydratases (GXGXXG((A/S))X((15-21))E((D))), were selected by alignment, and the spectral properties and kinetic parameters of their alanine-substituted variants were analyzed. Among the five variants, G29A, G31A, and E56A showed no interaction with FAD and exhibited no activity. These results indicate that G29, G31, and E56 are essential for FAD-binding.

Terminal olefin (1-alkene) biosynthesis by a novel p450 fatty acid decarboxylase from Jeotgalicoccus species.

Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleT(JE)), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleT(JE) is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or ß-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family.

Correlation of slime production investigated via three different methods in coagulase-negative staphylococci with crystal violet reaction and antimicrobial resistance.

This study investigated slime production by coagulase-negative staphylococci (CNS) using the standard tube (ST), Congo red agar (CRA) plate and Christensen's tube (CT) methods, and compared the results with those of the crystal violet reaction (CVR) test. The potential correlation between slime production and antimicrobial resistance was also evaluated. In total, 205 CNS strains were isolated from biological samples: 92 (44.9%) were shown to produce slime by the ST method; 96 (46.8%) by the CRA plate method; 90 (43.9%) by the CT method; and 89 (43.4%) strains were CVR positive. Eighty-three (40.5%) CNS strains were positive for slime production by the ST, CRA and CT methods. The findings of the ST, CRA and CT test methods were consistent with each other but were not related to CVR positivity. Based on the ST method, rates of antibiotic resistance to several antimicrobial agents were higher in slime-positive strains than in slime-negative strains and, in some cases, this was statistically significant.

[Search for lactate oxidase producer microorganisms].

Using the method of enrichment cultures, eight lactate oxidase producer strains of the fungus Geotrichum candidum were identified. The microorganisms were isolated from diverse specimens of fermented vegetables and manure. Variation in the content of glucose and lactate and the degree of aeration made it possible to attain lactate oxidase activities of up to 130-140 U per 11 grown medium containing microbial cells.

Microevolution of cytochrome bd oxidase in Staphylococci and its implication in resistance to respiratory toxins released by Pseudomonas.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and frequently coinfect the lungs of cystic fibrosis patients. P. aeruginosa secretes an arsenal of small respiratory inhibitors, like pyocyanin, hydrogen cyanide, or quinoline N-oxides, that may act against the commensal flora as well as host cells. Here, we show that with respect to their susceptibility to these respiratory inhibitors, staphylococcal species can be divided into two groups: the sensitive group, comprised of pathogenic species such as S. aureus and S. epidermidis, and the resistant group, represented by nonpathogenic species such as S. carnosus, S. piscifermentans, and S. gallinarum. The resistance in the latter group of species was due to cydAB genes that encode a pyocyanin- and cyanide-insensitive cytochrome bd quinol oxidase. By exchanging cydB in S. aureus with the S. carnosus-specific cydB, we could demonstrate that CydB determines resistance. The resistant or sensitive phenotype was based on structural alterations in CydB, which is part of CydAB, the cytochrome bd quinol oxidase. CydB represents a prime example of both microevolution and the asymmetric pattern of evolutionary change.