Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.Abbreviations C. parapsilosis Candida parapsilosis - S. cerevisiae Saccharomyces cerevisiae - C. utilis Candida utilis     

Characterization of the AINV gene and the encoded invertase from the dimorphic yeast Arxula adeninivorans

The invertase-encoding of AINV gene Arxula adeninivorans was isolated and characterized. The gene includes a coding sequence of 2700 bp encoding a putative 899 amino acid protein of 101.7 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of alpha-glucosidases from different sources. The gene activity is regulated by carbon source. In media supplemented with sucrose induction of the AINV gene and accumulation of the encoded invertase in the medium was observed. In addition the extracellular enzyme level is influenced by the morphological status of the organism, with mycelia secreting the enzyme in titres higher than those observed in budding yeasts. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AINV gene under control of the strong A. adeninivorans -derived TEF1 promoter. For both proteins a molecular mass of 600 kDa was determined, a pH optimum at pH 4.5 and a temperature optimum at 55 degrees C. The preferred substrates for the enzyme included the ss-D-fructofuranosides sucrose, inulin and raffinose. Only a weak enzyme activity was observed for the alpha-D-glucopyranosides maltotriose, maltose and isomaltose. Thus the invertase primarily is a ss-fructosidase and not an alpha-glucosidase as suggested by the homology to such enzymes.     

Candida parapsilosis fungemia in neonates: genotyping results suggest healthcare workers hands as source, and review of published studies

An outbreak of Candida parapsilosis fungemia involving 17 neonatal intensive care unit (NICU) patients was studied. There were 14 blood culture and nine colonizing isolates from other sites available. The hands of NICU healthcare workers (HCW) yielded eight isolates. Screening of the isolates by random amplified polymorphic DNA (RAPD) method showed only three profiles. Typing by restriction fragment length polymorphism (RFLP) revealed all blood isolates were RFLP subtype VII-1. Among the nine infant colonizing isolates, there were four different RFLP subtypes; four of the isolates were subtype VII-1. Seven of the eight isolates from HCW were RFLP subtype VII-1. The majority of infant colonizers were not found in the blood, suggesting a possible direct spread of the epidemic subtype VII-1 strain from HCW hands to infant blood. The source of the infant colonizing strains is unclear, but non-VII-1 strains may be largely of maternal origin and VII-1 strains from HCW. These findings reinforce prior studies that have implicated HCW hands as the source of nosocomial, including neonatal, fungemia.     

Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.     

Catabolism of 3-hydroxybenzoate by the gentisate pathway in Klebsiella pneumoniae M5a1

Growth of Klebsiella pneumoniae M5a1 on 3-hydroxybenzoate leads to the induction of 3-hydroxybenzoate monooxygenase, 2,5-dihydroxybenzoate dioxygenase, maleylpyruvate isomerase and fumarylpyruvate hydrolase. Growth in the presence of 2,5-dihydroxybenzoate also induces all of these enzymes including the 3-hydroxybenzoate monooxygenase which is not required for 2,5-dihydroxybenzoate catabolism. Mutants defective in 3-hydroxybenzoate monooxygenase fail to grow on 3-hydroxybenzoate but grow normally on 2,5-dihydroxybenzoate. Mutants lacking maleylpyruvate isomerase fail to grow on 3-hydroxybenzoate and 2,5-dihydroxybenzoate. Both kinds of mutants grow normally on 3,4-dihydroxybenzoate. Mutants defective in maleylpyruvate isomerase accumulate maleylpyruvate when exposed to 3-hydroxybenzoate and growth is inhibited. Secondary mutants that have additionally lost 3-hydroxybenzoate monooxygenase are no longer inhibited by the presence of 3-hydroxybenzoate. The 3-hydroxybenzoate monooxygenase gene (mhbM) and the maleylpyruvate isomerase gene (mhbI) are 100% co-transducible by P1 phage.     

Degradation and dehalogenation of monochlorophenols by the phenol-assimilating yeast Candida maltosa

The phenol-assimilating yeast Candida maltosa is able to degrade monochlorophenols but cannot grow on these substrates. 3- and 4-chlorophenol were broken down very rapidly by phenol-grown cells under the formation of 4-chlorocatechol, 5-chloropyrogallol and 4-carboxymethylenebut-2-en-4-olide with concomitant release of chloride.2-Chlorophenol was partially converted into cis,cis-2-chloromuconic acid via 3-chlorocatechol which was also obtained from 3-chlorophenol in low amounts. No further metabolites containing chloride were found.The dehalogenation step in the chlorophenol degradation is the cycloisomerization of the cis,cis-chloromuconic acid to 4-carboxymethylenebut-2-en-4-olide in the ortho fission pathway.Dedicated to prof.Dr. E. Bayer, Tübingen, on the occasion of his 65th birthday.     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

Enzymatic evidence for involvement of the methylmalonyl-CoA pathway in propionate oxidation by Syntrophobacter wolinii

Enzyme measurements were carried out with crude cell-free extracts of the propionate oxidizing coculture of Syntrophobacter wolinii and Desulfovibrio G11. Using cell-free extracts of a pure culture of Desulfovibrio G11 as a blank, most of the enzymes involved in the methylmalonyl-CoA pathway for propionate oxidation, including a propionyl-CoA: oxaloacetate transcarboxylase, were demonstrated in S. wolinii.     

Degradation kinetics of phenol by immobilized cells of Candida tropicalis in a fluidized bed reactor

Degradation kinetics of phenol by free and agar-entrapped cells of Candida tropicalis was studied in batch cultures. The initial phenol degradation rate achieved with free cells was higher than that obtained with immobilized cells, when phenol concentrations up to 1000 mg l^–1 were used. However, at higher phenol concentrations, the behaviour was quite different. The initial degradation rate of the immobilized yeast cells was about 10 times higher than that of the free cells, at a phenol concentration of 3500 mg l^–1. The semicontinuous and continuous degradation of phenol by immobilized yeast cells was also investigated in a multi-stage fluidized bed reactor. The highest phenol removal efficiencies and degradation rates as well as the lowest values of residual phenol and chemical oxygen demand were obtained in the semicontinuous culture when phenol concentrations up to 1560 mg l^–1 were used.     

Degradation of mandelate and 4-hydroxymandelate by Rhizobium leguminosarum biovar trifolii TA1

Rhizobium leguminosarum biovar trifolii TA1 grows on 4-hydroxymandelate and enzymes involved in its catabolism are inducible. Strain TA1 does not grown on mandelate or cis, cis-muconate, but spontaneous mutants capable of growth on these substrates were isolated. Enzymes involved in mandelate degradation were also inducible. The presence of intermediates of the mandelate and hydroxymandelate pathways resulted in a significant decrease in some of the enzymes involved in their degradation. Succinate and acetate, end products of the pathways, and glucose caused reductions in the levels of enzymes in the mandelate and hydroxymandelate pathways.     

A 9-year Study Comparing Risk Factors and the Outcome of Paediatric and Adults with Nosocomial Candidaemia

Although there are numerous studies of candidaemia in adults, data on paediatrics are still limited. The aim of this study was to compare risk factors, aetiology, therapy, and the outcome of nosocomial candidaemia among paediatric and adult patients in a large Brazilian tertiary hospital (1995–2003). During this period, 78 paediatrics and 113 adults were studied. Species other than Candida albicans caused 78.2% of episodes of candidaemia in paediatrics. Compared to adults, paediatrics received more frequently broad-spectrum antibiotics, vasopressors, blood transfusions, arterial catheter, chest tube, cardiothoracic surgery, mechanical ventilation, and parenteral nutrition. Candidaemia caused by Candida parapsilosis was more common in paediatrics, as was the isolation of Candida spp. from catheters. Amphotericin B treatment was more common in paediatrics. Mortality rate was higher in adults than in paediatrics with nosocomial candidaemia. We reinforce the necessity of continuous epidemiologic surveillance to follow the dynamics of candidaemia.     

Degradation of phenol and phenolic compounds by a defined denitrifying bacterial culture

Phenol, a major pollutant in several industrial waste waters is often used as a model compound for studies on biodegradation. This study investigated the anoxic degradation of phenol and other phenolic compounds by a defined mixed culture of Alcaligenes faecalis and Enterobacter species. The culture was capable of degrading high concentrations of phenol (up to 600 mg/l) under anoxic conditions in a simple minimal mineral medium at an initial cell mass of 8 mg/l. However, the lag phase in growth and phenol removal increased with increase in phenol concentration. Dissolved CO₂ was an absolute requirement for phenol degradation. In addition to nitrate, nitrite and oxygen could be used as electron acceptors. The kinetic constants, maximum specific growth rate _max; inhibition constant, K _i and saturation constant, K _s were determined to be 0.206 h^–1, 113 and 15 mg phenol/l respectively. p-Hydroxybenzoic acid was identified as an intermediate during phenol degradation. Apart from phenol, the culture utilized few other monocyclic aromatic compounds as growth substrates. The defined culture has remained stable with consistent phenol-degrading ability for more than 3 years and thus shows promise for its application in anoxic treatment of industrial waste waters containing phenolic compounds.     

Kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase

A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contribute to the binding of the anionic nitronate substrates to the enzymes. A linear correlation between the volumes of the inorganic anions and their effectiveness as competitive inhibitors of the enzymes was observed in a plot of pK_is versus the ionic volume of the anion with slopes of 0.041 ± 0.001 mM/ų and 0.027 ± 0.001 mM/ų for the H. mrakii and N. crassa enzymes, respectively. Aliphatic aldehydes were weak competitive inhibitors of the enzymes, with inhibition constants that are independent of their alkyl chain lengths. The reductive half reactions of H. mrakii nitronate monooxygenase with primary nitronates containing two to four carbon atoms all showed apparent K_d values of 5 mM. These results are consistent with the presence of an anion binding pocket in the active site of nitronate monooxygenase that interacts with the nitro group of the substrate, and suggest a minimal contribution of the hydrocarbon chain of the nitronates to the binding of the ligands to the enzyme.     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Ultrastructural and physico-chemical heterogeneities of yeast surfaces revealed by mapping lateral-friction and normal-adhesion forces using an atomic force microscope

Scanning force microscopy has been used to probe the surface of the emerging pathogenic yeast Candida parapsilosis, in order to get insight into its surface structure and properties at submicrometer scales. AFM friction images eventually show patches with a very strong contrast, showing high lateral interaction with the tip. Adhesion force measurement also reveals a high normal interaction with the tip, and patches show extraordinarily high pull off values. The tip eventually sticks completely at the center of the patches. While an extraordinarily high interaction is measured by the tip at those zones, topographic images show extraordinarily flat topography over those zones, both of which characteristics are consistent with a liquid-like area. High resolution friction images show those zones to be surrounded by microfibrillar structures, concentrically oriented, of a mean width of about 25 nm, structures that become progressively less defined as we move away from the center of the patches. No structure can be appreciated inside the zones of maximum contrast. Also some helical or ribbon-like structure can be resolved from friction images. There is not only an ordered disposition of the microfibrillar structures, but also the adhesion force increases radially in the direction towards the center of the patches. These structures responsible for the high adhesion are thought to be incipient-emerging budding zones. Microfibrillar structures are thought to represent the first steps of chitin biosynthesis and cell wall digestion, with chitin polymers being biosynthesized, associated with other macromolecules of the yeast cell wall. They can be also beta glucan helical structures, made visible in the zone of yeast division due to the action of autolysins. The observed gradient in surface adhesion and elastic properties correlates well with that expected from a biochemical point of view. The higher adhesion force measured could be either due to the different macromolecular nature of the patches, or to a mechanical adhesion effect due to the different plasticity of that zone. This work reveals the importance of taking into account the dynamic nature of the cell wall physico-chemical properties. Processes related to the normal cell-cycle, as division, can strongly alter the surface morphology and physico-chemical properties and cause important heterogeneities that might have a profound impact on the adhesion behavior of a single cell, which could not be detected by more macroscopic methods.     

Degradation pathway of an anthraquinone dye catalyzed by a unique peroxidase DyP from Thanatephorus cucumeris Dec 1

The reactants produced by action of a purified unique dye-decolorizing peroxidase, DyP, on a commercial anthraquinone dye, Reactive Blue 5, were investigated using electrospray ionization mass spectrometry (ESI-MS), thin-layer chromatography (TLC), and ¹H- and ¹³C- nuclear magnetic resonance (NMR). The results of ESI-MS analysis showed that phthalic acid, a Product 2 (molecular weight 472.5), and a Product 3 (molecular weight 301.5), were produced. Product 2 and Product 3 were generated by usual peroxidase reaction, whereas phthalic acid was generated by hydrolase- or oxygenase-catalyzed reaction. One potential associated product, o-aminobenzene sulfonic acid, was found to be converted to 2,2′-disulfonyl azobenzene by ESI-MS and NMR analyses. From these results, we propose, for the first time, the degradation pathway of an anthraquinone dye by the enzyme DyP.     

Disturbance of the secretory pathway inMicrasterias denticulata by tunicamycin and cyclopiazonic acid

Summary Both tunicamycin, an inhibitor of N-linked glycosylation of proteins, and cyclopiazonic acid, which inhibits the Ca²⁺-dependent ATPase in the ER, influence the secretory pathway at the ER level and lead to a cessation of cell growth inMicrasterias. Electron microscopical investigations reveal that the mode of action of the two inhibitors differs. While tunicamycin treatment results in a disintegration of the Golgi bodies into small vesicles, cyclopiazonic acid prevents products being supplied from the ER, resulting in the dilatation of ER cisternae and a reduction in the number of Golgi cisternae, combined with a loss of dictyosomal activity. The disturbed cell wall formation under tunicamycin indicates that N-linked glycosylation of proteins is required for normal cell growth inMicrasterias. Moreover, our studies reveal that changes in cytoplasmic free calcium concentration, as a consequence of ATPase inhibition in the ER by cyclopiazonic acid, may inhibit wall material secretion by interrupting the normal ER-dictyosome association.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum - TM tunicamycin     

Interaction between sulphur mobilisation proteins SufB and SufC: evidence for an iron-sulphur cluster biogenesis pathway in the apicoplast of Plasmodium falciparum

The plastid of Plasmodium falciparum, the apicoplast, performs metabolic functions essential to the parasite. Various reactions in the plastid require the assembly of [Fe-S] prosthetic groups on participating proteins as well as the reductant activity of ferredoxin that is converted from its apo-form by the assembly of [Fe-S] clusters inside the apicoplast. The [Fe-S] assembly pathway involving sulphur mobilising Suf proteins has been predicted to function in the apicoplast with one component (PfSufB) encoded by the plastid genome itself. We demonstrate the ATPase activity of recombinant P. falciparum nuclear-encoded SufC and its localisation in the apicoplast. Further, an internal region of apicoplast SufB was used to detect PfSufB-PfSufC interaction in vitro; co-elution of SufB from parasite lysate with recombinant PfSufC on an affinity column also indicated an interaction of the two proteins. As a departure from bacterial SufB and similar to reported plant plastid SufB, apicoplast SufB exhibited ATPase activity, suggesting the evolution of specialised functions in the plastid counterparts. Our results provide experimental evidence for an active Suf pathway in the Plasmodium apicoplast.     

Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth

A biosynthetic pathway for rosmarinic acid is proposed. This pathway is deduced from studies of the enzymes detectable in preparations from suspension cells of Coleus blumei. Phenylalanine is transformed to 4-coumaroyl-CoA by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11) and hydroxycinnamic acid:CoA ligase (EC 6.2.1.12). Tyrosine is metabolized to 4-hydroxyphenyllactate by tyrosine aminotransferase (EC 2.6.1.5) and hydroxyphenylpyruvate reductase. The ester can be formed from 4-coumaroyl-CoA and 4-hydroxyphenyllactate by the catalytic activity of rosmarinic acid synthase with concomitant release of CoA. Microsomal hydroxylase activities introduce the hydroxyl groups at positions 3 and 3 of the aromatic rings of the ester 4-coumaroyl-4-hydroxyphenyllactate giving rise to rosmarinic acid.Abbreviations Caf-pHPL caffeoyl-4-hydroxyphenyllactate - DHPL 3,4-dihydroxyphenyllactic acid - pC-DHPL 4-coumaryl-3,4-dihydroxyphenyllactate - pC-pHPL 4-coumaryl-4-hydroxyphenyllactate - pHPL 4-hydroxyphenyllactic acid - RA rosmarinic acid The financial support of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged.     

Induced neuroma formation and target muscle perturbation in the giant fiber pathway of the Drosophila temperature-sensitive mutant shibire

Summary The temperature-sensitive mutation shibire (shi) in Drosophila melanogaster is thought to disrupt membrane recycling processes, including endocytotic vesicle pinch-off. This mutation can perturb the development of nerves and muscles of the adult escape response. After exposure to a heat pulse (6 h at 30° C) at 20 h of pupal development, adults have abnormal flight muscles. Wing depressor muscles (DLM) are reduced in number from the normal six to one or two fibers, and are composed of enlarged fibers that appear to represent fiber fusion; large spaces devoid of muscle fibers suggested fiber deletion. The normal five motor axons are present in the peripheral nerve PDMN near the ganglion. However, while some motor axons pass dorsally to the extant fibers, other motor axons lacking end targets pass into an abnormal posterior branch and terminate in a neuroma, i.e., a tangle of axons and glia without muscle target tissue. Hemisynapses are common in axons of the proximal PDMN and within the neuroma, but they are rarely seen in control (no heat pulse) shi or wild-type flies. All surviving muscle fibers are innervated; no muscle tissue exists without innervation. Fibrillar fine structure and neuromuscular synapses appear normal. Fused fibers have dual innervation, suggesting correct and specific matching of target tissue and motor axons. Motor axons lacking target fibers do not innervate erroneous targets but instead terminate in the neuroma. These results suggest developmental constraints and rules, which may contribute to the orderly, stereotyped development in the normal flight system. The nature of the anomalies inducible in the flight motor system in shi flies implies that membrane recycling events at about 20 h of pupal development are critical to the formation of the normal adult nerve-muscle pattern for DLM flight muscles.