Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions

Dictyostelium cells that lack the myoB isoform were previously shown to exhibit reduced efficiencies of phagocytosis and chemotactic aggregation ("streaming") and to crawl at about half the speed of wild- type cells. Of the four other Dictyostelium myosin I isoforms identified to date, myoC and myoD are the most similar to myoB in terms of tail domain sequence. Furthermore, we show here that myoC, like myoB and myoD, is concentrated in actin-rich cortical regions like the leading edge of migrating cells. To look for evidence of functional overlap between these isoforms, we analyzed myoB, myoC, and myoD single mutants, myoB/myoD double mutants, and myoB/myoC/myoD triple mutants, which were created using a combination of gene targeting techniques and constitutive expression of antisense RNA. With regard to the speed of locomoting, aggregation-stage cells, of the three single mutants, only the myoB mutant was significantly slower. Moreover, double and triple mutants were only slightly slower than the myoB single mutant. Consistent with this, the protein level of myoB alone rises dramatically during early development, suggesting that a special demand is placed on this one isoform when cells become highly motile. We also found, however, that the absolute amount of myoB protein in aggregation- stage cells is much higher than that for myoC and myoD, suggesting that what appears to be a case of nonoverlapping function could be the result of large differences in the amounts of functionally overlapping isoforms. Streaming assays also suggest that myoC plays a significant role in some aspect of motility other than cell speed. With regard to phagocytosis, both myoB and myoC single mutants exhibited significant reductions in initial rate, suggesting that these two isoforms perform nonredundant roles in supporting the phagocytic process. In triple mutants these defects were not additive, however. Finally, because double and triple mutants exhibited significant and progressive decreases in doubling times, we also measured the kinetics of fluid phase endocytic flux (uptake, transit time, efflux). Not only do all three isoforms contribute to this process, but their contributions are synergistic. While these results, when taken together, refute the simple notion that these three "classic" myosin I isoforms perform exclusively identical functions, they do reveal that all three share in supporting at least one cellular process (endocytosis), and they identify several other processes (motility, streaming, and phagocytosis) that are supported to a significant extent by either individual isoforms or various combinations of them.     

F-actin Distribution of Dictyostelium Myosin I Double Mutants

The roles of the myosin I class of mechanoenzymes have been investigated by single and double gene knockout studies in the amoeba Dictyostelium discoideum. Cells lacking different myosin I pairs (myoA^-/myoB^-, myoB^-/myoC^-, and myoA^-/myoC^-) were examined with respect to their cytoskeletal organization. F-actin localization by rhodamine-phalloidin staining of cells indicates that the myoA^-/myoB^-, myoB^-/myoC^-, and myoA^-/myoC^- cells appear to redistribute their F-actin more slowly than wild type cells upon adhesion to a substrate. These studies suggest that Dictyostelium myoA, myoB, and myoC may have overlapping roles in maintaining the integrity or organization of the cortical membrane cytoskeleton.     

The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains.

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH(2)-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.     

A myosin I is involved in membrane recycling from early endosomes

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin-myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA(-)/B(-), and myoB(-) cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA(-)/B(-) cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.     

Myosin I Overexpression Impairs Cell Migration

Dictyostelium myoB, a member of the myosin I family of motor proteins, is important for controlling the formation and retraction of membrane projections by the cell's actin cortex (Novak, K.D., M.D. Peterson, M.C. Reedy, and M.A. Titus. 1995. J. Cell Biol. 131:1205–1221). Mutants that express a three- to sevenfold excess of myoB (myoB⁺ cells) were generated to further analyze the role of myosin I in these processes. The myoB⁺ cells move with an instantaneous velocity that is 35% of the wild-type rate and exhibit a 6–8-h delay in initiation of aggregation when placed under starvation conditions. The myoB⁺ cells complete the developmental cycle after an extended period of time, but they form fewer fruiting bodies that appear to be small and abnormal. The myoB⁺ cells are also deficient in their ability both to form distinct F-actin filled projections such as crowns and to become elongate and polarized. This defect can be attributed to the presence of at least threefold more myoB at the cortex of the myoB⁺ cells. In contrast, threefold overexpression of a truncated myoB that lacks the src homology 3 (SH3) domain (myoB/SH3⁻ cells) or myoB in which the consensus heavy chain phosphorylation site was mutated to an alanine (S332A-myoB) does not disturb normal cellular function. However, there is an increased concentration of myoB in the cortex of the myoB/SH3⁻ and S332A-myoB cells comparable to that found in the myoB⁺ cells. These results suggest that excess full-length cortical myoB prevents the formation of the actin-filled extensions required for locomotion by increasing the tension of the F-actin cytoskeleton and/ or retracting projections before they can fully extend. They also demonstrate a role for the phosphorylation site and SH3 domain in mediating the in vivo activity of myosin I.     

Redundancy in the microfilament system: abnormal development of Dictyostelium cells lacking two F-actin cross-linking proteins.

We generated by gene disruption Dictyostelium cells that lacked both the F-actin cross-linking proteins, alpha-actinin and gelation factor. Several major cell functions, such as growth, chemotaxis, phagocytosis, and pinocytosis, were apparently unaltered. However, in all double mutants, development was greatly impaired. After formation of aggregates, cells were very rarely able to form fruiting bodies. This ability was rescued when mutant and wild-type strains were mixed in a ratio of 70 to 30. The developmental program in the mutant was not arrested, since the expression pattern of early and late genes remained unchanged. Development of the mutant was rendered normal when a functional alpha-actinin gene was introduced and expressed, showing the morphogenetic defect to be due to the absence of the two F-actin cross-linking proteins. These findings suggest the existence of a functional network allowing mutual complementation of certain actin-binding proteins.     

myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth

We have identified and cloned a novel essential myosin I in Aspergillus nidulans called myoA. The 1,249-amino acid predicted polypeptide encoded by myoA is most similar to the amoeboid myosins I. Using affinity-purified antibodies against the unique myosin I carboxyl terminus, we have determined that MYOA is enriched at growing hyphal tips. Disruption of myoA by homologous recombination resulted in a diploid strain heterozygous for the myoA gene disruption. We can recover haploids with an intact myoA gene from these strains, but never haploids that are myoA disrupted. These data indicated that myoA encodes an essential myosin I, and this has allowed us to use a unique approach to studying myosin I function. We have developed conditionally null myoA strains in which myoA expression is regulated by the alcA alcohol dehydrogenase promoter. A conditionally lethal strain germinated on inducing medium grows as wild type, displaying polarized growth by apical extension. However, growth of the same myoA mutant strain on repressing medium results in enlarged cells incapable of hyphal extension, and these cells eventually die. Under repressing conditions, this strain also displays reduced levels of secreted acid phosphatase. The mutant phenotype indicates that myoA plays a critical role in polarized growth and secretion.     

Localization of wild type and mutant class I myosin proteins in Aspergillus nidulans using GFP-fusion proteins

We have examined the distribution of MYOA, the class I myosin protein of the filamentous fungus Aspergillus nidulans, as a GFP fusion protein. Wild type GFP-MYOA expressed from the myoA promoter is able to rescue a conditional myoA null mutant. Growth of a strain expressing GFP-MYOA as the only class I myosin was approximately 50% that of a control strain, demonstrating that the fusion protein retains substantial myosin function. The distribution of the wild type GFP-MYOA fusion is enriched in growing hyphal tips and at sites of septum formation. In addition, we find that GFP-MYOA is also found in patches at the cell cortex. We have also investigated the effects of deletion or truncation mutations in the tail domain on MYOA localization. Mutant GFP-MYOA fusions that lacked either the C-terminal SH3 or a portion of the C-terminal proline-rich domain had subcellular distributions like wild type MYOA, consistent with their ability to complement a myoA null mutant. In contrast, mutants lacking all of the C-terminal proline-rich domain or the TH-1-like domain were mainly localized diffusely throughout the cytoplasm, but could less frequently be found in patches, and were unable to complement a myoA null mutant. The GFP-MYOA DeltaIQ mutant was localized into large bright fluorescent patches in the cytoplasm. This mutant protein was subsequently found to be insoluble.     

Dynamin A, Myosin IB and Abp1 couple phagosome maturation to F-actin binding

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.     

Specific sterols required for the internalization step of endocytosis in yeast

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.     

Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain- deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.     

The unconventional myosin encoded by the myoA gene plays a role in Dictyostelium motility.

The myoA gene of Dictyostelium is a member of a gene family of unconventional myosins. The myosin Is share homologous head and basic domains, but the myoA gene product lacks the glycine-, proline-, alanine-rich and src homology 3 domains typical of several of the other myosin Is. A mutant strain of Dictyostelium lacking a functional myoA gene was produced by gene targeting, and the motility of this strain in buffer and a spatial gradient of the chemoattractant cyclic AMP was analyzed by computer-assisted methods. The myoA- cells have a normal elongate morphology in buffer but exhibit a decrease in the instantaneous velocity of cellular translocation, an increase in the frequency of lateral pseudopod formation, and an increase in turning. In a spatial gradient, in which the frequency of pseudopod formation is depressed, myoA- cells exhibit positive chemotaxis but still turn several times more frequently than control cells. These results demonstrate that the other members of the unconventional myosin family do not fully compensate for the loss of functional myoA gene product. Surprisingly, the phenotype of the myoA- strain closely resembles that of the myoB- strain, suggesting that both play a role in the frequency of pseudopod formation and turning during cellular translocation.     

Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.     

Genetic analysis of membrane differentiation in Paramecium. Freeze- fracture study of the trichocyst cycle in wild-type and mutant strains

Using a series of mutants of Paramecium tetraurelia, we demonstrate, for the first time, changes in the internal structure of the cell membrane, as revealed by freeze-fracture, that correspond to specific single gene mutations. On the plasma membrane of Paramecium circular arrays of particles mark the sites of attachment of the tips of the intracellular secretory organelles-trichocysts. In wild-type paramecia, where attached trichocysts can be expelled by exocytosis under various stimuli, the plasma membrane array is composed of a double outer ring of particles (300 nm in diameter) and inside the ring a central rosette (fusion rosette) of particles (76 nm in diameter). Mutant nd9, characterized by a thermosensitive ability to discharge trichocysts, shows the same organization in cells grown at the permissive temperature (18 degrees C), while in cells grown at the nonpermissive temperature (27 degrees C) the rosette is missing. In mutant tam 8, characterized by normal but unattached trichocysts, and in mutant tl, completely devoid of trichocysts, no rosette is formed and the outer rings always show a modified configuration called "parentheses", also found in wild-type and in nd9 (18 degrees C) cells. From this comparison between wild type and mutants, we conclude: (a) that the formation of parentheses is a primary differentiation of the plasma membrane, independent of the presence of trichocysts, while the secondary transformation of parentheses into circular arrays and the formation of the rosette are triggered by interaction between trichocysts and plasma membranes; and (b) that the formation of the rosette is a prerequisite for trichocyst exocytosis.     

The Dictyostelium essential light chain is required for myosin function.

A Dictyostelium mutant (7-11) that expresses less than 0.5% of wild-type levels of the myosin essential light chain (EMLC) has been created by overexpression of antisense RNA. Cells from 7-11 contain wild-type levels of the myosin heavy chain (MHC) and regulatory light chain (RMLC). Myosin isolated from 7-11 cells consists of the MHC with the RMLC associated in reduced stoichiometry, and binds to purified actin in an ATP-sensitive fashion. Purified 7-11 myosin displays calcium-activated ATPase activity with a Vmax about 15%-25% of that of wild type, and a Km for ATP of 27 +/- 5 microM versus 83 +/- 30 microM for wild type. At actin concentrations as high as 17 microM, 7-11 myosin displays greatly reduced actin-activated ATPase activity. Phenotypically, 7-11 cells resemble MHC mutants, growing poorly in suspension and becoming large and multinucleate. When starved for multicellular development, 7-11 cells take several hours longer than wild-type cells to aggregate. Although multicellular aggregates eventually form, they fail to develop further. The cells are also unable to cap receptors in response to Con A treatment. Since cells expressing the EMLC are phenotypically similar to MHC null mutants, the EMLC appears necessary for myosin function, at least in part because it is required for normal actin-activated ATPase activity.     

Cytokinesis is defective in Dictyostelium mutants with altered phagocytic recognition, adhesion, and vegetative cell cohesion properties

Mutants that have been selected for defects in phagocytic recognition, adhesion, and vegetative cell-cell cohesion were found to be larger and more highly multinucleate than their parent strain. This defect is associated with the complex mutant phenotype of these mutants since revertants of the mutants coordinately acquire the wild-type phenotype for all of the defects. The larger size and multinuclearity were due to a high frequency of failure of cytokinesis in cells of wild-type size. This was shown by purifying the small cells in mutant populations and observing their growth and cell division. The mutant phenotype is more penetrant during axenic growth. Most of the mutants are not multinucleate when grown on bacteria. Recently, new mutants have been isolated that are also multinucleate when grown on bacteria by a strong selection procedure for non-adhesion to tissue culture dishes. The pleiotropic mutant phenotype and the greater penetrance of the mutant phenotype in axenic culture can be explained by hypothesizing a deficiency in a membrane component of the actomyosin motor that is involved in all of the processes defective in the mutants.     

One-eyed pinhead regulates cell motility independent of Squint/Cyclops signaling

In vertebrates, EGF-CFC factors are essential for Nodal signaling. Here, we show that the zygotic function of one-eyed pinhead, the zebrafish EGF-CFC factor, is necessary for cell movement throughout the blastoderm of the early embryo. During the blastula and gastrula stages, mutant cells are more cohesive and migrate slower than wild-type cells. Chimeric analysis reveals that these early motility defects are cell-autonomous; later, one-eyed pinhead mutant cells have a cell-autonomous tendency to acquire ectodermal rather than mesendodermal fates. Moreover, wild-type cells transplanted into the axial region of mutant hosts tend to form isolated aggregates of notochord tissue adjacent to the mutant notochord. Upon misexpressing the Nodal-like ligand Activin in whole embryos, which rescues aspects of the mutant phenotype, cell behavior retains the one-eyed pinhead motility phenotype. However, in squint;cyclops double mutants, which lack Nodal function and possess a more severe phenotype than zygotic one-eyed pinhead mutants, cells of the dorsal margin exhibit a marked tendency to widely disperse rather than cohere together. Elsewhere in the double mutants, for cells of the blastoderm and for rare cells of the gastrula that involute into the hypoblast, motility appears wild-type. Notably, cells at the animal pole, which are not under direct regulation by the Nodal pathway, behave normal in squint;cyclops mutants but exhibit defective motility in one-eyed pinhead mutants. We conclude that, in addition to a role in Nodal signaling, One-eyed pinhead is required for aspects of cell movement, possibly by regulating cell adhesion.     

The adaptor protein Bam32 regulates Rac1 activation and actin remodeling through a phosphorylation-dependent mechanism

The B cell adaptor molecule of 32 kDa (Bam32) is an adaptor that links the B cell antigen receptor (BCR) to ERK and JNK activation and ultimately to mitogenesis. After BCR cross-linking, Bam32 is recruited to the plasma membrane and accumulates within F-actin-rich membrane ruffles. Bam32 contains one Src homology 2 and one pleckstrin homology domain and is phosphorylated at a single site, tyrosine 139. To define the function of Bam32 in membrane-proximal signaling events, we established human B cell lines overexpressing wild-type or mutant Bam32 proteins. The basal level of F-actin increased in cells expressing wild-type or myristoylated Bam32 but decreased in cells expressing either an Src homology-2 or Tyr-139 Bam32 mutant. Overexpression of wild-type Bam32 also affected BCR-induced actin remodeling, which was visualized as increases in F-actin-rich membrane ruffles. In contrast, Bam32 mutants largely blocked the BCR-induced increase in cellular F-actin. The positive and negative effects of Bam32 variants on F-actin levels were closely mirrored by their effects on the activation of the GTPase Rac1, which is known to regulate actin remodeling in lymphocytes. Bam32-deficient DT40 B cells showed decreased Rac1 activation and a failure of Rac1 to co-localize with the BCR, whereas cells overexpressing Bam32 had increased constitutive Rac1 activation. These results suggest that Bam32 regulates the cytoskeleton through Rac1. Bam32 variants also affected downstream signaling to JNK in a manner similar to that of Rac1, suggesting that the effect of Bam32 on JNK activation may be at least partially mediated through Rac1. Our results demonstrate a novel phosphorylation-dependent function of Bam32 in regulating Rac1 activation and actin remodeling.     

Pyruvate formate-lyase is not essential for nitrate respiration by Escherichia coli

Abstract Defined deletion mutants of Escherichia coli defective for the synthesis of pyruvate formate-lyase (PFL) or pyruvate dehydrogenase (PDH) were analysed in regards their growth in batch culture and their enzyme levels under fermentative and nitrate respiratory conditions. A pfl mutant proved not to be completely auxotrophic for acetate when grown anaerobically in glucose minimal medium. In contrast, a pfl aceEF double mutant exhibited an absolute requirement for acetate, indicating that PDH is the source of acetyl-CoA in the pfl mutant. Growth of both pfl and aceEF single mutants under nitrate respiratory conditions was essentially indistinguishable from the wild-type. Thus, either PFL or PDH can be used to catabolise pyruvate in nitrate-respiring cells. The activities of PFL and PDH measured after growth with nitrate are commensurate with this proposal.