BACKGROUND: In C. elegans there are two well-defined TGFbeta-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFbeta signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. RESULTS: kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase) family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. CONCLUSION: kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFbeta outputs, such as tail morphogenesis.

The nematode Caenorhabditis elegans has no kidney per se, yet ``the worm'' has proved to be an excellent model to study renal-related issues, including tubulogenesis of the excretory canal, membrane transport and ion channel function, and human genetic diseases including autosomal dominant polycystic kidney disease (ADPKD). The goal of this review is to explain how C. elegans has provided insight into cilia development, cilia function, and human cystic kidney diseases.

Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.

Nephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease in children and young adults. In Chlamydomonas reinhardtii, Caenorhabditis elegans, and mammals, the NPHP1 and NPHP4 gene products nephrocystin-1 and nephrocystin-4 localize to basal bodies or ciliary transition zones (TZs), but their function in this location remains unknown. We show here that loss of C. elegans NPHP-1 and NPHP-4 from TZs is tolerated in developing cilia but causes changes in localization of specific ciliary components and a broad range of subtle axonemal ultrastructural defects. In amphid channel cilia, nphp-4 mutations cause B tubule defects that further disrupt intraflagellar transport (IFT). We propose that NPHP-1 and NPHP-4 act globally at the TZ to regulate ciliary access of the IFT machinery, axonemal structural components, and signaling molecules, and that perturbing this balance results in cell type-specific phenotypes.

The formation of pseudouridine (Psi) from uridine is post-transcriptional and catalysed by pseudouridine synthases, several of which have been characterized from eukaryotes. Pseudouridine synthase 1 (Pus1p) has been well characterized from yeast and mice. In yeast, Pus1p has been shown to have dual substrate specificity, modifying uridines in tRNAs and at position 44 in U2 small nuclear RNA (U2 snRNA). In order to study the in vivo activity of a metazoan Pus1p, a knockout of the gene coding for the homologue of Pus1p in Caenorhabditis elegans was obtained. The deletion encompasses the first two putative exons and includes the essential aspartate that is required for activity in truA pseudouridine synthases. The locations of most modified nucleotides on small RNAs in C. elegans are not known, and the positions of Psi were determined on four tRNAs and U2 snRNA. The uridine at position 27 of tRNA(Val) (AAC), a putative Pus1p-modification site, was converted into Psi in the wild-type worms, but the tRNA(Val) (AAC) from mutant worms lacked the modification. Psi formation at positions 13, 32, 38 and 39, all of which should be modified by other pseudouridine synthases, was not affected by the loss of Pus1p. The absence of Pus1p in C. elegans had no effect on the modification of U2 snRNA in vivo, even though worm U2 snRNA has a Psi at position 45 (the equivalent of yeast U2 snRNA position 44) and at four other positions. This result was unexpected, given the known dual specificity of yeast Pus1p.

Bone morphogenetic protein (BMP) pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-10 as a positive extracellular regulator of BMP-like receptor signaling. SMA-10 acts genetically in a BMP-like (Sma/Mab) pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-10 and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane proteins. SMA-10 is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-10(lf) animals with the Drosophila ortholog lambik, showing that SMA-10 physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.

Perturbation of the catalytic inorganic core (Mn4Ca1OxCly) of the photosystem II-water-oxidizing complex (PSII-WOC) isolated from spinach is examined by substitution of Ca2+ with cadmium(II) during core assembly. Cd2+ inhibits the yield of reconstitution of O-2-evolution activity, called photoactivation, starting from the free inorganic cofactors and the cofactor-depleted apo-WOC-PSII complex. Ca2+ affinity increases following photooxidation of the first Mn2+ to Mn3+ bound to the 'high-affinity' site. Ca2+ binding occurs in the dark and is the slowest overall step of photoactivation (IM1/IM*(1) -> step). Cd2+ competitively blocks the binding of Ca2+ to its functional site with 10-to 30-fold higher affinity, but does not influence the binding of Mn2+ to its high-affinity site. By contrast, even 10-fold higher concentrations of Cd2+ have no effect on O-2-evolution activity in intact PSII-WOC. Paradoxically, Cd2+ both inhibits photoactivation yield, while accelerating the rate of photoassembly of active centres 10-fold relative to Ca2+. Cd2+ increases the kinetic stability of the photooxidized Mn3+ assembly intermediate(s) by twofold (mean lifetime for dark decay). The rate data provide evidence that Cd2+ binding following photooxidation of the first Mn3+, IM1/IM*(1), causes three outcomes: (i) a longer intermediate lifetime that slows IM1 decay to IM0 by charge recombination, (ii) 10-fold higher probability of attaining the degrees of freedom (either or both cofactor and protein d.f.) needed to bind and photooxidize the remaining 3 Mn2+ that form the functional cluster, and (iii) increased lability of Cd2+ following Mn-4 cluster assembly results in (re) exchange of Cd2+ by Ca2+ which restores active O-2-evolving centres. Prior EPR spectroscopic data provide evidence for an oxo-bridged assembly intermediate, Mn3+ (mu-O2-) Ca2+, for IM*(1). We postulate an analogous inhibited intermediate with Cd2+ replacing Ca2+.

Synaptic connections undergo a dynamic process of stabilization or elimination during development, and this process is thought to be critical in memory and learning and in establishing the specificity of synaptic connections. The type II calcium- and calmodulin-dependent protein kinase (CaMKII) has been proposed to be pivotal in regulating synaptic strength and in maturation of synapses during development. Here we describe how CaMKII regulates the formation of central glutamatergic synapses in Caenorhabditis elegans. During larval development, the density of ventral nerve cord synapses containing the GLR-1 glutamate receptor is held constant despite marked changes in neurite length. The coupling of synapse number to neurite length requires both CaMKII and voltage-gated calcium channels. CaMKII regulates GLR-1 by at least two distinct mechanisms: regulating transport of GLR-1 from cell bodies to neurites; and regulating the addition or maintenance of GLR-1 to postsynaptic elements.

Protein-protein interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation. Interactions between catabolite gene activator protein (CAP) and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA. Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending). All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP. Here we use the thermodynamically rigorous technique of fluorescence polarization to show that CAP interacts with RNA polymerase in solution in the absence of promoter DNA (KD,app = 2.8 x 10(-7) M), whereas [Ala158]CAP, a mutant of CAP specifically defective in transcription activation, does not.

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Mycobacterium tuberculosis is arguably the world's most successful infectious agent because of its ability to control its own cell growth within the host. Bacterial growth rate is closely coupled to rRNA transcription, which in E. coli is regulated through DksA and (p)ppGpp. The mechanisms of rRNA transcriptional control in mycobacteria, which lack DksA, are undefined. Here we identify CarD as an essential mycobacterial protein that controls rRNA transcription. Loss of CarD is lethal for mycobacteria in culture and during infection of mice. CarD depletion leads to sensitivity to killing by oxidative stress, starvation, and DNA damage, accompanied by failure to reduce rRNA transcription. CarD can functionally replace DksA for stringent control of rRNA transcription, even though CarD associates with a different site on RNA polymerase. These findings highlight a distinct molecular mechanism for regulating rRNA transcription in mycobacteria that is critical for M. tuberculosis pathogenesis.

BACKGROUND: In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19-25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana, mutations in CARPEL FACTORY (CAF), a Dicer homolog, and those in a novel gene, HEN1, result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism.RESULTS: To investigate the potential functions of CAF and HEN1 in miRNA metabolism, we aimed to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. We have isolated 11 miRNAs, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes.CONCLUSIONS: miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis, operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis, and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development.

Cilia serve as sensory devices in a diversity of organisms and their defects contribute to many human diseases. In primary cilia of kidney cells, the transient receptor potential polycystin (TRPP) channels polycystin-1 (PC-1) and polycystin-2 (PC-2) act as a mechanosensitive channel, with defects resulting in autosomal dominant polycystic kidney disease. In sensory cilia of Caenorhabditis elegans male-specific neurons, the TRPPs LOV-1 and PKD-2 are required for mating behavior. The mechanisms regulating TRPP ciliary localization and function are largely unknown. We identified the regulatory subunit of the serine-threonine casein kinase II (CK2) as a binding partner of LOV-1 and human PC-1. CK2 and the calcineurin phosphatase TAX-6 modulate male mating behavior and PKD-2 ciliary localization. The phospho-defective mutant PKD-2(S534A) localizes to cilia, whereas a phospho-mimetic PKD-2(S534D) mutant is largely absent from cilia. Calcineurin is required for PKD-2 ciliary localization, but is not essential for ciliary gene expression, ciliogenesis, or localization of cilium structural components. This unanticipated function of calcineurin may be important for regulating ciliary protein localization. A dynamic phosphorylation-dephosphorylation cycle may represent a mechanism for modulating TRPP activity, cellular sensation, and ciliary protein localization.

Recently determined structures of the Escherichia coli catabolite activator protein (CAP) in complex with DNA, and in complex with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) and DNA, have yielded insights into how CAP binds DNA and activates transcription. Comparison of multiple structures of CAP-DNA complexes has revealed the contributions of direct and indirect readout to DNA binding by CAP. The structure of the CAP-alphaCTD-DNA complex has provided the first structural description of interactions between a transcription activator and its functional target within the general transcription machinery. Using the structure of the CAP-alphaCTD-DNA complex, the structure of an RNA polymerase-DNA complex, and restraints from biophysical, biochemical and genetic experiments, it has been possible to construct detailed three-dimensional models of intact class I and class II transcription activation complexes.

It has been suggested that the catabolite gene activator protein (CAP) uses an "acidic activating region" transcription activation mechanism and that Glu171 of CAP is the critical amino acid of the "acidic activating region" of CAP (Irwin, N., and Ptashne, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8315-8319). In this paper, we show, contrary to the previously published report, that substitution of Glu171 of CAP fails to result in a specific defect in transcription activation at the lac promoter. Furthermore, in this paper, we show that substitution of each other negatively charged amino acid of CAP that is solvent-accessible in the CAP-DNA complex fails to result in a specific defect in transcription activation at the lac promoter. We conclude that CAP does not use an acidic activating region transcription activation mechanism in transcription activation at the lac promoter.

Kline et al. (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E. coli. We have examined this phenomenon in detail, utilizing fusions between the structural gene for beta-galactosidase and the promoters for the araBAD, araE, and araFG operons. We confirm that IAA potently stimulates transcription from the araBAD promoter. The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons. However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP. Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene.

Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcL(S)) produced tobacco(Rst) transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (L(s)S(t)). The tobacco(Rst) plants required CO(2) (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K(m), for CO(2) and CO(2)/O(2) selectivity of the L(s)S(t) enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf L(s)S(t) content were sufficient, tobacco(Rst) plants grew to maturity without CO(2) supplementation. When grown under a high pCO(2), the tobacco(Rst) seedlings grew slower than tobacco and exhibited unique growth phenotypes: Juvenile plants formed clusters of 10 to 20 structurally simple oblanceolate leaves, developed multiple apical meristems, and the mature leaves displayed marginal curling and dimpling. Depending on developmental stage, the L(s)S(t) content in tobacco(Rst) leaves was 4- to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO(2) and growth illumination CO(2) assimilation in mature tobacco(Rst) leaves remained limited by Rubisco activity and its rate (approximately 11 micromol m(-2) s(-1)) was half that of tobacco controls. (35)S-methionine labeling showed the stability of assembled L(s)S(t) was similar to tobacco Rubisco and measurements of light transient CO(2) assimilation rates showed L(s)S(t) was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobacco(Rst) growth primarily stem from reduced rbcL(S) mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted L(s)S(t) synthesis.

After the onset of gastrulation, the Drosophila germband undergoes a morphological change in which its length along the anterior-posterior axis increases over two-and-a-half fold while its width along the dorsal-ventral axis simultaneously narrows. The behavior of individual cells during germband extension was investigated by epi-illumination and time-lapse video microscopy of living embryos. Cells intercalate between their dorsal and ventral neighbors during extension, increasing the number of cells along the anterior-posterior axis while decreasing the number of cells along the dorsal-ventral axis. Mutations that reduce segmental subdivision of the embryo along the anterior-posterior axis decrease both germband extension and its associated cell intercalation. In contrast, cell intercalation and germband extension are still detected in embryos that lack dorsal-ventral polarity. Characterization of germband extension and cell intercalation in mutant embryos with altered segmentation gene expression indicates that these processes are regionally autonomous and are dependent upon the establishment of striped expression patterns for certain pair-rule genes. Based on these observations, we propose a model for germband extension in which cell intercalation results from the establishment of adhesive differences between stripes of cells by pair-rule genes.

Appendage formation in insects and vertebrates depends upon signals from both the anterior-posterior and dorsal-ventral (DV) axes. In Drosophila, wing formation is organized symmetrically around the DV boundary of the growing wing imaginal disc and requires interactions between dorsal and ventral cells. Compartmentalization of the wing disc, dorsal cell behavior, and the expression of two dorsally expressed putative signaling molecules, fringe (fng) and Serrate (Ser), are regulated by the apterous selector gene. Here, we demonstrate that fng and Ser have distinct roles in a novel cell recognition and signal induction process. fng serves as a boundary-determining molecule such that Ser is induced wherever cells expressing fng and cells not expressing fng are juxtaposed. Ser in turn triggers the expression of genes involved in wing growth and patterning on both sides of the DV boundary.

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization following allotetraploidization.

Notch proteins are receptors for a conserved signaling pathway that affects numerous cell fate decisions. We found that in Drosophila, Protein O-fucosyltransferase 1 (OFUT1), an enzyme that glycosylates epidermal growth factor-like domains of Notch, also has a distinct Notch chaperone activity. OFUT1 is an endoplasmic reticulum protein, and its localization was essential for function in vivo. OFUT1 could bind to Notch, was required for the trafficking of wild-type Notch out of the endoplasmic reticulum, and could partially rescue defects in secretion and ligand binding associated with Notch point mutations. This ability of OFUT1 to facilitate folding of Notch did not require its fucosyltransferase activity. Thus, a glycosyltransferase can bind its substrate in the endoplasmic reticulum to facilitate normal folding.

The Drosophila Dachsous and Fat proteins function as ligand and receptor, respectively, for an intercellular signaling pathway that regulates Hippo signaling and planar cell polarity. Although gene-targeted mutations in two mammalian Fat genes have been described, whether mammals have a Fat signaling pathway equivalent to that in Drosophila, and what its biological functions might be, have remained unclear. Here, we describe a gene-targeted mutation in a murine Dachsous homolog, Dchs1. Analysis of the phenotypes of Dchs1 mutant mice and comparisons with Fat4 mutant mice identify requirements for these genes in multiple organs, including the ear, kidney, skeleton, intestine, heart and lung. Dchs1 and Fat4 single mutants and Dchs1 Fat4 double mutants have similar phenotypes throughout the body. In some cases, these phenotypes suggest that Dchs1-Fat4 signaling influences planar cell polarity. In addition to the appearance of cysts in newborn kidneys, we also identify and characterize a requirement for Dchs1 and Fat4 in growth, branching and cell survival during early kidney development. Dchs1 and Fat4 are predominantly expressed in mesenchymal cells in multiple organs, and mutation of either gene increases protein staining for the other. Our analysis implies that Dchs1 and Fat4 function as a ligand-receptor pair during murine development, and identifies novel requirements for Dchs1-Fat4 signaling in multiple organs.

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase, which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-CoA. A nifJ knock-out mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to wild type (WT) under light-dark cycling. This is attributed to an increase in the quantum yield of PSII charge separation as measured by photosynthetic electron turnover efficiency using fast repetition rate fluorometry (Fv/Fm). During autofermentation the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT, H2 production in vivo was 1.3-fold lower than WT. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with resulting loss of reductant and a 3-fold decrease in H2 production by nifJ cells compared to WT. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin because it decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD+ ratio revealed that the reductant generated by PFOR contributing to the first phase of H2 production was not in equilibrium with bulk NADH/NAD+, while the second phase corresponded to the equilibrium NADH-mediated process.