Unravelling HetC as a peptidase-based ABC exporter driving functional cell differentiation in the cyanobacterium Nostoc PCC 7120.

The export of peptides or proteins is essential for a variety of important functions in bacteria. Among the diverse protein-translocation systems, peptidase-containing ABC transporters (PCAT) are involved in the maturation and export of quorum-sensing or antimicrobial peptides in Gram-positive bacteria and of toxins in Gram-negative organisms. In the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120, the protein HetC is essential for the differentiation of functional heterocysts, which are micro-oxic and non-dividing cells specialized in atmospheric nitrogen fixation. HetC shows similarities to PCAT systems, but whether it actually acts as a peptidase-based exporter remains to be established. In this study, we show that the N-terminal part of HetC, encompassing the peptidase domain, displays a cysteine-type protease activity. The conserved catalytic residues conserved in this family of proteases are essential for the proteolytic activity of HetC and the differentiation of heterocysts. Furthermore, we show that the catalytic residue of the ATPase domain of HetC is also essential for cell differentiation. Interestingly, HetC has a cyclic nucleotide-binding domain at its N-terminus which can bind ppGpp in vitro and which is required for its function in vivo. Our results indicate that HetC is a peculiar PCAT that might be regulated by ppGpp to potentially facilitate the export of a signaling peptide essential for cell differentiation, thereby broadening the scope of PCAT role in Gram-negative bacteria.IMPORTANCEBacteria have a great capacity to adapt to various environmental and physiological conditions; it is widely accepted that their ability to produce extracellular molecules contributes greatly to their fitness. Exported molecules are used for a variety of purposes ranging from communication to adjust cellular physiology, to the production of toxins that bacteria secrete to fight for their ecological niche. They use export machineries for this purpose, the most common of which energize transport by hydrolysis of adenosine triphosphate. Here, we demonstrate that such a mechanism is involved in cell differentiation in the filamentous cyanobacterium Nostoc PCC 7120. The HetC protein belongs to the ATP-binding cassette transporter superfamily and presumably ensures the maturation of a yet unknown substrate during export. These results open interesting perspectives on cellular signaling pathways involving the export of regulatory peptides, which will broaden our knowledge of how these bacteria use two cell types to conciliate photosynthesis and nitrogen fixation.

HetL, HetR and PatS form a reaction-diffusion system to control pattern formation in the cyanobacterium nostoc PCC 7120.

Local activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns. A number of them involve the production by the developing cell of an inhibitory morphogen, but how this cell becomes immune to self-inhibition is rather unknown. Under combined nitrogen starvation, the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of one heterocyst every 10-12 vegetative cells. Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogens, diffusion of which establishes the differentiation pattern. Here, we show that HetR interacts with HetL at the same interface as PatS, and that this interaction is necessary to suppress inhibition and to differentiate heterocysts. hetL expression is induced under nitrogen-starvation and is activated by HetR, suggesting that HetL provides immunity to the heterocyst. This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum.

Cyanobacteria are the only bacteria on Earth able to draw their energy directly from the sun in the same way that plants do. In addition, some strains are able to 'fix' the nitrogen present in the atmosphere: they can extract this gas and transform it into nitrogen-based compounds necessary for life. However, both processes cannot happen in a given cell at the same time. A strain of cyanobacteria called Nostoc PCC 7120 can organise itself into long filaments of interconnected cells. Under certain conditions, one in every ten cells stops drawing its energy from the sun, and starts fixing atmospheric nitrogen instead. Exactly how the bacteria are able to 'count to ten' and organize themselves in such a precise pattern is still unclear. Cells can communicate and establish patterns by exchanging molecular signals that switch on and off certain cell programs. For instance, a protein called HetR turns on the genetic program that allows cyanobacteria to fix nitrogen; on the other hand, a signal known as PatS binds to HetR and turns it off. Cells starting to specialise in fixing nitrogen produce both HetR and PatS, with the latter diffusing in surrounding cells and preventing them from extracting nitrogen. However, it remained unclear how the nitrogen-fixing cell could ignore its own PatS signal and keep its HetR signal active. HetL ­ another protein produced by the future nitrogen-fixing cell ­ could potentially play this role, but how it acts was unknown. Here, Xu et al. show that HetL cannot diffuse from one cell to the other, and that it binds to HetR at the same place than PatS does. When both PatS and HetL are present, they compete to attach to HetR, which stops PatS from turning off HetR and deactivating the nitrogen-fixing program. Understanding how cyanobacteria fix nitrogen could help to develop new types of natural fertiliser. More generally, dissecting how these simple organisms can create patterns could help to grasp how patterning emerges in more complex creatures.

Transcription termination controls prophage maintenance in Escherichia coli genomes.

Prophages represent a large fraction of prokaryotic genomes and often provide new functions to their hosts, in particular virulence and fitness. How prokaryotic cells maintain such gene providers is central for understanding bacterial genome evolution by horizontal transfer. Prophage excision occurs through site-specific recombination mediated by a prophage-encoded integrase. In addition, a recombination directionality factor (or excisionase) directs the reaction toward excision and prevents the phage genome from being reintegrated. In this work, we describe the role of the transcription termination factor Rho in prophage maintenance through control of the synthesis of transcripts that mediate recombination directionality factor expression and, thus, excisive recombination. We show that Rho inhibition by bicyclomycin allows for the expression of prophage genes that lead to excisive recombination. Thus, besides its role in the silencing of horizontally acquired genes, Rho also maintains lysogeny of defective and functional prophages.

Chaperone-assisted excisive recombination, a solitary role for DnaJ (Hsp40) chaperone in lysogeny escape.

Temperate bacteriophage lytic development is intrinsically related to the stress response in particular at the DNA replication and virion maturation steps. Alternatively, temperate phages become lysogenic and integrate their genome into the host chromosome. Under stressful conditions, the prophage resumes a lytic development program, and the phage DNA is excised before being replicated. The KplE1 defective prophage of Escherichia coli K12 constitutes a model system because it is fully competent for integrative as well as excisive recombination and presents an atypical recombination module, which is conserved in various phage genomes. In this work, we identified the host-encoded stress-responsive molecular chaperone DnaJ (Hsp40) as an active participant in KplE1 prophage excision. We first show that the recombination directionality factor TorI of KplE1 specifically interacts with DnaJ. In addition, we found that DnaJ dramatically enhances both TorI binding to its DNA target and excisive recombination in vitro. Remarkably, such stimulatory effect by DnaJ was performed independently of its DnaK chaperone partner and did not require a functional DnaJ J-domain. Taken together, our results underline a novel and unsuspected functional interaction between the generic host stress-regulated chaperone and temperate bacteriophage lysogenic development.

Tight regulation of the intS gene of the KplE1 prophage: a new paradigm for integrase gene regulation.

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excessive recombination are discussed.

Protein binding sites involved in the assembly of the KplE1 prophage intasome.

The organization of the recombination regions of the KplE1 prophage in Escherichia coli K12 differs from that observed in the lambda prophage. Indeed, the binding sites characterized for the IntS integrase, the TorI recombination directionality factor (RDF) and the integration host factor (IHF) vary in number, spacing and orientation on the attL and attR regions. In this paper, we performed site-directed mutagenesis of the recombination sites to decipher if all sites are essential for the site-specific recombination reaction and how the KplE1 intasome is assembled. We also show that TorI and IntS form oligomers that are stabilized in the presence of their target DNA. Moreover, we found that IHF is the only nucleoid associated protein (NAP) involved in KplE1 recombination, although it is dispensable. This is consistent with the presence of only one functional IHF site on attR and none on attL.

Introduction of methionines in the gas channel makes [NiFe] hydrogenase aero-tolerant.

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

Experimental approaches to kinetics of gas diffusion in hydrogenase.

Hydrogenases, which catalyze H(2) to H(+) conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H(2) toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.

The zinc finger protein Ynr046w is plurifunctional and a component of the eRF1 methyltransferase in yeast.

Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 A resolution. It comprises a zinc-binding domain built from both the N- and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three alpha-helices. The active methyltransferase is the heterodimer Ydr140w.Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold.

Molecular basis for bacterial class I release factor methylation by PrmC.

Class I release factors bind to ribosomes in response to stop codons and trigger peptidyl-tRNA hydrolysis at the P site. Prokaryotic and eukaryotic RFs share one motif: a GGQ tripeptide positioned in a loop at the end of a stem region that interacts with the ribosomal peptidyl transferase center. The glutamine side chain of this motif is specifically methylated in both prokaryotes and eukaryotes. Methylation in E. coli is due to PrmC and results in strong stimulation of peptide chain release. We have solved the crystal structure of the complex between E. coli RF1 and PrmC bound to the methyl donor product AdoHCy. Both the GGQ domain (domain 3) and the central region (domains 2 and 4) of RF1 interact with PrmC. Structural and mutagenic data indicate a compact conformation of RF1 that is unlike its conformation when it is bound to the ribosome but is similar to the crystal structure of the protein alone.

The glutamine residue of the conserved GGQ motif in Saccharomyces cerevisiae release factor eRF1 is methylated by the product of the YDR140w gene.

Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to release factor (RF) activity in both kingdoms. In bacteria, the Gln residue of the motif in RF1 and RF2 is modified to N(5)-methyl-Gln by the S-adenosyl l-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae release factor eRF1, the first time that N(5)-methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial release factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl l-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1.eRF3.GTP.

The essential role of the invariant GGQ motif in the function and stability in vivo of bacterial release factors RF1 and RF2.

Release factors RF1 and RF2 are required in bacteria for the cleavage of peptidyl-tRNA. A single sequence motif, GGQ, is conserved in all eubacterial, archaebacterial and eukaryotic release factors and may mimic the CCA end of tRNA, although the position of the motif in the crystal structures of human eRF1 and Escherichia coli RF2 is strikingly different. Mutations have been introduced at each of the three conserved positions. Changing the Gln residue to Ala or Glu allowed the factors to retain about 22% of tetrapeptide release activity in vitro, but these mutants could not complement thermosensitive RF mutants in vivo. None of several mutants with altered Gly residues retained activity in vivo or in vitro. Many GGQ mutants were poorly expressed and are presumably unstable; many were also toxic to the cell. The toxic mutant factors or their degradation products may bind to ribosomes inhibiting the action of the normal factor. These data are consistent with a common role for the GGQ motif in bacterial and eukaryotic release factors, despite strong divergence in primary, secondary and tertiary structure, but are difficult to reconcile with the hypothesis that the amide nitrogen of the Gln plays a vital role in peptidyl-tRNA hydrolysis.

The hemK gene in Escherichia coli encodes the N(5)-glutamine methyltransferase that modifies peptide release factors.

Class 1 peptide release factors (RFs) in Escherichia coli are N(5)-methylated on the glutamine residue of the universally conserved GGQ motif. One other protein alone has been shown to contain N(5)-methylglutamine: E.coli ribosomal protein L3. We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2. HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro. hemK is immediately downstream of and co-expressed with prfA. Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1. The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N(5)-methylation of Gln252. Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2. HemK and YfcB are the first identified methyltransferases modifying glutamine, and are widely distributed in nature.