Patterning, morphogenesis, and neurogenesis of zebrafish cranial sensory placodes.

Peripheral sensory organs and ganglia found in the vertebrate head arise during embryonic development from distinct ectodermal thickenings, called cranial sensory placodes (adenohypophyseal, olfactory, lens, trigeminal, epibranchial, and otic). A series of patterning events leads to the establishment of these placodes. Subsequently, these placodes undergo specific morphogenetic movements and cell-type specification in order to shape the final placodal derivatives and to produce differentiated cell types necessary for their function. In this chapter, we will focus on recent studies in the zebrafish that have advanced our understanding of cranial sensory placode development. We will summarize the signaling events and their molecular effectors guiding the formation of the so-called preplacodal region, and the subsequent subdivision of this region along the anteroposterior axis that gives rise to specific placode identities as well as those controlling morphogenesis and neurogenesis. Finally, we will highlight the approaches used in zebrafish that have been established to precisely label cell populations, to follow their development, and/or to characterize cell fates within a specific placode.

Three calcium-sensitive genes, fus, brd3 and wdr5, are highly expressed in neural and renal territories during amphibian development.

Numerous Ca(2+) signaling events have been associated with early development of vertebrate embryo, from fertilization to organogenesis. In Xenopus laevis, Ca(2+) signals are key regulators in the earliest steps of the nervous system development. If neural determination is one of the best-characterized examples of the role of Ca(2+) during embryogenesis, increasing literature supports a determining role of organogenesis and differentiation. In blastula the cells of the presumptive ectoderm (animal caps) are pluripotent and can be induced toward neural fate with an intracellular increase of free Ca(2+) triggered by caffeine. To identify genes that are transcribed early upon Ca(2+) stimuli and involved in neural determination, we have constructed a subtractive cDNA library between neuralized and non-neuralized animal caps. Here we present the expression pattern of three new Ca(2+)-sensitive genes: fus (fused in sarcoma), brd3 (bromodomain containing 3) and wdr5 (WD repeat domain 5) as they all represent potential regulators of the transcriptional machinery. Using in situ hybridization we illustrated the spatial expression pattern of fus, brd3 and wdr5 during early developmental stages of Xenopus embryos. Strikingly, their domains of expression are not restricted to neural territories. They all share a specific expression throughout renal organogenesis which has been found to rely also on Ca(2+) signaling. This therefore highlights the key function of Ca(2+) target genes in specific territories during early development. We propose that Ca(2+) signaling through modulation of fus, brd3 and wdr5 expressions can control the transcription machinery to achieve proper embryogenesis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Isolation, free-living capacities, and genome structure of "Candidatus Glomeribacter gigasporarum," the endocellular bacterium of the mycorrhizal fungus Gigaspora margarita.

"Candidatus Glomeribacter gigasporarum" is an endocellular beta-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita. We established a protocol to isolate "Ca. Glomeribacter gigasporarum" from its host which allowed us to carry out morphological, physiological, and genomic investigations on purified bacteria. They are rod shaped, with a cell wall typical of gram-negative bacteria and a cytoplasm rich in ribosomes, and they present no flagella or pili. Isolated bacteria could not be grown in any of the 19 culture media tested, but they could be kept alive for up to 4 weeks. PCR-based investigations of purified DNA from isolated bacteria did not confirm the presence of all genes previously assigned to "Ca. Glomeribacter gigasporarum." In particular, the presence of nif genes could not be detected. Pulsed-field gel electrophoresis analyses allowed us to estimate the genome size of "Ca. Glomeribacter gigasporarum" to approximately 1.4 Mb with a ca. 750-kb chromosome and a 600- to 650-kb plasmid. This is the smallest genome known for a beta-proteobacterium. Such small genome sizes are typically found in endocellular bacteria living permanently in their host. Altogether, our data suggest that "Ca. Glomeribacter gigasporarum" is an ancient obligate endocellular bacterium of the AM fungus G. margarita.

Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021.

Sinorhizobium meliloti is an alpha-proteobacterium that forms agronomically important N(2)-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid/peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.

Mutation in the ntrR gene, a member of the vap gene family, increases the symbiotic efficiency of Sinorhizobium meliloti.

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the "ntrPR operon," which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti.

BACKGROUND: Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown. RESULTS: We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis. CONCLUSIONS: Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.

[Neural determination in Xenopus laevis embryos: control of early neural gene expression by calcium]. / La détermination neurale dans l'embryon de Xenopus laevis: contrôle de l'expression des gènes neuraux précoces par le calcium.

In amphibian embryos the central nervous system derives from the dorsal region of the ectoderm. Molecular studies led to the formulation of the "neural default model" in which neural development is under the inhibitory control of members of the BMP family. These growth factors also act as epidermis inducers. The neural fate is revealed by factors secreted by the Spemann Organizer such as noggin, chordin, follistatin, Xnr3 and cerberus which act by blocking BMP signalling. We propose a new model for neural cell determination in which a signalling pathway controlled by an increase in intracellular calcium suppresses the epidermis fate and activates the neural fate instead. This increase in calcium is due to an influx through calcium channels of the L-type, expressed in ectodermal cells during gastrulation. The possible involvement of a calcium-dependent phosphatase (calcineurin) to inhibit the epidermis fate and a calcium-calmodulin kinase (CaMkinase II) which activates the neural fate is discussed.

Identification of Sinorhizobium meliloti genes regulated during symbiosis.

RNA fingerprinting by arbitrarily primed PCR was used to isolate Sinorhizobium meliloti genes regulated during the symbiotic interaction with alfalfa (Medicago sativa). Sixteen partial cDNAs were isolated whose corresponding genes were differentially expressed between symbiotic and free-living conditions. Thirteen sequences corresponded to genes up-regulated during symbiosis, whereas three were instead repressed during establishment of the symbiotic interaction. Seven cDNAs corresponded to known or predicted nif and fix genes. Four presented high sequence similarity with genes not yet identified in S. meliloti, including genes encoding a component of the pyruvate dehydrogenase complex, a cell surface protein component, a copper transporter, and an argininosuccinate lyase. Finally, five cDNAs did not exhibit any similarity with sequences present in databases. A detailed expression analysis of the nine non-nif-fix genes provided evidence for an unexpected variety of regulatory patterns, most of which have not been described so far.

High-resolution physical map of the pSymb megaplasmid and comparison of the three replicons of Sinorhizobium meliloti strain 1021.

A high-resolution physical map of the larger megaplasmid (pSymb) of Sinorhizobium meliloti strain 1021 has been constructed by using BAC libraries and an original two-step PCR screening method. This method, previously used to map both the chromosome and the smaller megaplasmid (pSyma), allowed us to position over the genome a total of 842 markers with an average density of one marker every 8.3 kb. In addition, we used BLASTX and PRODOM analysis to predict a function for a number of STSs. This work led to the discovery of several interesting loci and to a comparison of the genetic information carried by each replicon. The two main results emerging from this study are (i) a biased distribution of housekeeping genes, mainly detected on chromosome, and (ii) the presence of an unexpected number of transporters, mainly belonging to the ABC superfamily. These are broadly distributed across the whole genome, but particularly found on pSymb.

Symbiotic induction of pyruvate dehydrogenase genes from Sinorhizobium meliloti.

Genes coding for components of the pyruvate dehydrogenase (PDH) multienzyme complex (PDHc) from Sinorhizobium meliloti, the alfalfa symbiont, have been isolated on the basis of their high expression in symbiotic bacteria. The Elp component, PDH, is encoded by two genes, pdhAalpha (1,047 bp) and pdhAbeta (1,383 bp), a situation encountered in the alpha-proteobacteria Rickettsia prowazekii and Zymomonas mobilis as well as in some gram-positive bacteria and in mitochondria. pdhAalpha and pdhAbeta precede pdhB (1,344 bp), which encodes the E2p component, dihydrolipoamide acetyltransferase, of the PDHc. No gene encoding the E3 component, lipoamide dehydrogenase, was found in the immediate vicinity of pdhA and pdhB genes. pdhAalpha, pdhAbeta and pdhB likely constitute an operon. Here, we provide evidence that pdhA expression is induced in the symbiotic stage, compared with free-living conditions. We demonstrate that symbiotic expression of pdhA genes does not depend on the fix LJ regulatory cascade that regulates nitrogen fixation and respiration gene expression in symbiotic S. meliloti cells. Induction of pdhA expression could be obtained under free-living conditions upon the addition of pyruvate to the culture medium. Induction by pyruvate and symbiotic activation of pdh gene expression take place at the same promoter.

Inhibition of the FixL sensor kinase by the FixT protein in Sinorhizobium meliloti.

Nitrogen fixation in symbiotic rhizobia is subject to multiple levels of gene regulation. In Sinorhizobium meliloti, the alfalfa symbiont, the FixLJ two-component regulatory system plays a major role in inducing nitrogen fixation and respiration gene expression in response to the low ambient O(2) concentration of the nodule. Here we report on the mode of action of the FixT protein, a recently identified repressor of nitrogen fixation gene expression in S. meliloti. First, we provide evidence that FixT prevents transcription of the intermediate key regulatory genes nifA and fixK by counteracting the activity of the FixLJ two-component system under otherwise inducing microoxic conditions. Second, we demonstrate that FixT acts as an inhibitor of the sensor hemoprotein kinase FixL, preventing the production or the accumulation of its phosphorylated form. FixT is thus a new example of a regulatory protein that blocks signal transduction in two-component systems at the level of the sensor kinase.

Negative autoregulation of the Rhizobium meliloti fixK gene is indirect and requires a newly identified regulator, FixT.

fixK genes are crp/fnr homologues that have been discovered in diverse Rhizobium spp., in which they are usually essential for symbiotic nitrogen fixation. One recurrent function of fixK genes in rhizobia is to activate the transcription of operons required for respiration in the microoxic environment of the nodule. In a similar manner to its Escherichia coli crp and fnr homologues, R. meliloti fixK regulates its own expression negatively. However, we demonstrate here that fixK negative autoregulation is not direct and, instead, involves a newly identified gene, fixT, the expression of which depends on fixK. Inactivation of fixT resulted in derepression of fixK expression under free-living microoxic conditions. Furthermore, constitutively expressed fixT strongly repressed fixK-lacZ expression in the absence of a functional fixK gene. Several lines of evidence indicate that fixT is active via its protein product FixT. FixT does not resemble any protein present in databases so far. Nodules induced by a fixT mutant were Fix+, thus demonstrating that fixT is not essential for symbiotic nitrogen fixation.

Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule.

The symbiotic pattern of expression of Rhizobium meliloti N2-fixation genes is tightly coupled with the histological organization of the alfalfa root nodule and thus is under developmental control. N2-fixation gene expression is induced very sharply at a particular zone of the nodule called interzone II-III that precedes the zone where N2 fixation takes place. We show here that this coupling can be disrupted, hereby resulting in ectopic expression of N2-fixation genes in the prefixing zone II of the nodule. Uncoupling was obtained either by using a R. meliloti strain in which a mutation rendered N2-fixation gene expression constitutive with respect to oxygen in free-living bacterial cultures or by placing nodules induced by a wild-type R. meliloti strain in a microoxic environment. These results implicate oxygen as a key determinant of the symbiotic pattern of N2-fixation gene expression.

Phosphorylation of the Rhizobium meliloti FixJ protein induces its binding to a compound regulatory region at the fixK promoter.

The FixJ protein is a member of the regulator class of two-component systems involved in the transcriptional activation of nitrogen fixation genes in Rhizobium meliloti. Phosphorylation of FixJ was previously demonstrated to dramatically enhance its transcriptional activity at the nifA and fixK promoters. Here we show that the isolated carboxyl-terminal domain of FixJ, FixJC, binds the fixK promoter, whereas binding of the full-length FixJ protein requires its phosphorylation. By analyzing the DNase I and Exonuclease III protection patterns of the wild-type and a mutant fixK promoter, we have identified two overlapping binding regions for both phosphorylated FixJ and FixJC. A higher affinity region is located between positions -69 and -44 relative to the transcription start site, and a lower affinity region, between positions -57 and -31, overlaps the -35 region of the promoter.

FixL of Rhizobium meliloti enhances the transcriptional activity of a mutant FixJD54N protein by phosphorylation of an alternate residue.

In Rhizobium meliloti, transcription of nitrogen fixation genes is induced in oxygen-depleted conditions under the control of the two-component regulatory system FixLJ. FixJ is a transcriptional activator whose activity is dramatically enhanced by phosphorylation, whereas FixL is a hemoprotein kinase that controls the level of phosphorylated FixJ in response to oxygen availability. We have found that a mutant FixJ protein, FixJD54N, in which the presumed site of phosphorylation (aspartate 54) was changed to an asparagine, is strongly affected for phosphorylation by FixL and is not detectably phosphorylated from the low-molecular-weight phosphate donor, acetyl-phosphate. Unexpectedly, FixL strongly enhances the transcriptional activity of the FixJD54N protein both in vivo and in vitro. We present evidence that FixJD54N transcriptional activity is enhanced by phosphorylation of an alternate residue in a reaction that requires FixL and ATP and is not affected by oxygen. We also demonstrate the key role of Asp-54 of FixJ in oxygen signal transduction.

Oxygen control in Rhizobium.

Rhizobia are gram-negative bacteria with two distinct habitats: the soil rhizosphere in which they have a saprophytic and, usually, aerobic life and a plant ecological niche, the legume nodule, which constitutes a microoxic environment compatible with the operation of the nitrogen reducing enzyme nitrogenase. The purpose of this review is to summarize the present knowledge of the changes induced in these bacteria when shifting to a microoxic environment. Oxygen concentration regulates the expression of two major metabolic pathways: energy conservation by respiratory chains and nitrogen fixation. After reviewing the genetic data on these metabolic pathways and their response to oxygen we will put special emphasis on the regulatory molecules which are involved in the control of gene expression. We will show that, although homologous regulatory molecules allow response to oxygen in different species, they are assembled in various combinations resulting in a variable regulatory coupling between genes for microaerobic respiration and nitrogen fixation genes. The significance of coordinated regulation of genes not essential for nitrogen fixation with nitrogen fixation genes will also be discussed.

Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes.

Oxygen concentration regulates the expression of nitrogen fixation genes in the symbiotic bacterium Rhizobium meliloti. We demonstrate that two proteins, FixL and FixJ, that belong to the two-component family of regulatory proteins are necessary and sufficient for oxygen-regulated in vitro transcription of the two key regulatory genes, nifA and fixK. We show directly that FixJ is a transcriptional activator, working in conjunction with the RNA polymerase sigma 70 holoenzyme. Addition of FixL122, a soluble form of the sensor FixL protein, to the transcription assay enhanced FixJ transcriptional activity in response to low oxygen concentration. This enhancement of FixJ activity was correlated with FixJ phosphorylation.

Modular structure of the FixL protein of Rhizobium meliloti.

FixL protein of Rhizobium meliloti is a haemo-protein kinase which activates the transcription of nifA and fixK genes via the transcriptional activator protein FixJ under microaerobic conditions. FixL and FixJ proteins belong to the family of two-component regulatory systems for which primary sequence data predicts a modular structure. We showed, using Escherichia coli as heterologous host, that FixL indeed has a modular structure. The amino-terminal hydrophobic domain is dispensable for the oxygen-regulated activity of FixL in vivo. The central cytoplasmic non-conserved domain is necessary for the oxygen-sensing function of FixL whereas it is not necessary for the activation of FixJ by FixL. We propose that, under aerobic conditions, the central domain represses the activating function associated with the carboxy-terminal conserved domain.

Molecular genetic analysis of the Rhizobium meliloti fixK promoter: identification of sequences involved in positive and negative regulation.

Transcription of the Rhizobium meliloti fixK gene is induced in symbiotic and microaerobic growth conditions by the FixL/FixJ modulator/effector pair. Transcription of fixK is also negatively autoregulated. By 5' deletion analysis, the involvement in negative regulation of a DNA region between -514 and -450 with respect to the transcription start was demonstrated. Site-directed mutagenesis allowed us to show that a sequence homologous to the binding site of the Escherichia coli Fnr protein, centred at position -487, participates in this effect. However, deletion or mutagenesis of this Fnr-like sequence does not completely eliminate FixK-dependent repression, which suggests that either an additional DNA region is involved in negative regulation or that it is mediated at the level of fixLJ transcription. Deletion analysis also allowed the definition of a DNA region involved in FixJ-mediated activation of the fixK promoter, between -79 and -42. Different point mutations in the -60, -45 and -35 regions were shown to affect promoter activity. In some cases, the activity of mutant promoters could be partly or fully restored by increasing the expression of the fixLJ regulatory genes, in an E. coli strain harbouring a plasmid with fixLJ under the control of an inducible (p-tac) promoter.

The phosphorylated form of the enhancer-binding protein NTRC has an ATPase activity that is essential for activation of transcription.

The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription in response to limitation of combined nitrogen. NTRC activates transcription by catalyzing formation of open complexes by RNA polymerase (sigma 54 holoenzyme form) in an ATP-dependent reaction. To catalyze open complex formation, NTRC must be phosphorylated. We show that phosphorylated NTRC has an ATPase activity, and we present biochemical and genetic evidence that NTRC must hydrolyze ATP to catalyze open complex formation. It is likely that all activators of sigma 54 holoenzyme have an ATPase activity.