Shingo Nozaki

An activator for pyruvoyl-dependent l-aspartate α-decarboxylase is conserved in a small group of the γ-proteobacteria including Escherichia coli.

In bacteria, β‐alanine is formed via the action of l‐aspartate α‐decarboxylase (PanD) which is one of the small class of pyruvoyl‐dependent enzymes. The pyruvoyl cofactor in these enzymes is formed via the intramolecular rearrangement of a serine residue in the peptide backbone leading to chain cleavage and formation of the covalently‐bound cofactor from the serine residue. This reaction was previously thought to be uncatalysed. Here we show that in Escherichia coli, PanD is activated by the putative acetyltransferase YhhK, subsequently termed PanZ. Activation of PanD both in vivo and in vitro is PanZ‐dependent. PanZ binds to PanD, and we demonstrate that a PanZ(N45A) site‐directed mutant is unable to enhance cleavage of the proenzyme PanD despite retaining affinity for PanD. This suggests that the putative acetyltransferases domain of PanZ may be responsible for activation to enhance the processing of PanD. Although panD is conserved among most bacteria, the panZ gene is conserved only in E. coli‐related enterobacterial species including Shigella, Salmonella, Klebsiella and Yersinia. These bacteria are found predominantly in the gut flora where pantothenate is abundant and regulation of PanD by PanZ allows these organisms to closely regulate production of β‐alanine and hence pantothenate in response to metabolic demand.

Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in DeltadatA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.

Synchronous Activation of Cell Division by Light or Temperature Stimuli in the Dimorphic Yeast Schizosaccharomyces japonicus

ABSTRACT Many fungi respond to light and regulate fungal development and behavior. A blue light-activated complex has been identified in Neurospora crassa as the product of the wc-1 and wc-2 genes. Orthologs of WC-1 and WC-2 have hitherto been found only in filamentous fungi and not in yeast, with the exception of the basidiomycete pathogenic yeast Cryptococcus. Here, we report that the fission yeast Schizosaccharomyces japonicus responds to blue light depending on Wcs1 and Wcs2, orthologs of components of the WC complex. Surprisingly, those of ascomycete S. japonicus are more closely related to those of the basidiomycete. S. japonicus reversibly changes from yeast to hyphae in response to environmental stresses. After incubation at 30°C, a colony of yeast was formed, and then hyphal cells extended from the periphery of the colony. When light cycles were applied, distinct dark- and bright-colored hyphal cell stripes were formed because the growing hyphal cells had synchronously activated cytokinesis. In addition, temperature cycles of 30°C for 12 h and 35°C for 12 h or of 25°C for 12 h and 30°C for 12 h during incubation in the dark induced a response in the hyphal cells similar to that of light. The stripe formation of the temperature cycles was independent of the wcs genes. Both light and temperature, which are daily external cues, have the same effect on growing hyphal cells. A dual sensing mechanism of external cues allows organisms to adapt to daily changes of environmental alteration.

The Structure of the PanD/PanZ Protein Complex Reveals Negative Feedback Regulation of Pantothenate Biosynthesis by Coenzyme A

Summary Coenzyme A (CoA) is an ubiquitous and essential cofactor, synthesized from the precursor pantothenate. Vitamin biosynthetic pathways are normally tightly regulated, including the pathway from pantothenate to CoA. However, no regulation of pantothenate biosynthesis has been identified. We have recently described an additional component in the pantothenate biosynthetic pathway, PanZ, which promotes the activation of the zymogen, PanD, to form aspartate α-decarboxylase (ADC) in a CoA-dependent manner. Here we report the structure of PanZ in complex with PanD, which reveals the structural basis for the CoA dependence of this interaction and activation. In addition, we show that PanZ acts as a CoA-dependent inhibitor of ADC catalysis. This inhibitory effect can effectively regulate the biosynthetic pathway to pantothenate, and thereby also regulate CoA biosynthesis. This represents a previously unobserved mode of metabolic regulation whereby a cofactor-utilizing protein negatively regulates the biosynthesis of the same cofactor.

Formation of a heterooctameric complex between aspartate α-decarboxylase and its cognate activating factor, PanZ, is CoA-dependent.

Highlights ► PanZ is required for formation of the pyruvoyl-cofactor in PanD (ADC) in vivo. ► PanZ and PanD interact with nanomolar affinity. ► Interaction of PanZ and PanD is dependent upon coenzyme A. ► PanZ and PanD form a heterooctameric complex which binds four molecules of CoA.

The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD–PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan)

The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of β-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked β-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.

Exonuclease III (XthA) enforces in vivo DNA cloning of Escherichia coli to create cohesive ends

Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15-40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA-cloning technology. However, the molecular mechanism by which E. coli accomplishes such cloning is still unknown. In this study, we provide evidence that the in vivo cloning of E. coli is independent of both RecA and RecET recombinase, but is dependent on XthA, a 3’ to 5’ exonuclease. Here, in vivo cloning of E. coli by XthA is referred to as iVEC (in vivo E. coli cloning). Next, we show that the iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA-cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC. Importance Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, in vitro recombination of DNA fragments effectively joins multiple DNA fragments in place of the canonical method. Interestingly, E. coli can take up linear double-stranded vectors, insert DNA fragments and assemble them in vivo. The in vivo cloning have realized a high level of usability comparable to that by in vitro recombination reaction, since now it is only necessary to introduce PCR products into E. coli for the in vivo cloning. However, the mechanism of in vivo cloning is highly controversial. Here we clarified the fundamental mechanism underlying in vivo cloning of E. coli and also constructed an E. coli strain that was optimized for in vivo cloning.

The Ras1-Cdc42 pathway is involved in hyphal development of Schizosaccharomyces japonicus

Dimorphic yeasts transform into filamentous cells or hyphae in response to environmental cues. The mechanisms for the hyphal transition of dimorphic yeasts have mainly been studied in Candida albicans, an opportunistic human fungal pathogen. The Ras1-MAPK pathway is a major signal transduction pathway for hyphal transition in C. albicans. Recently, the non-pathogenic dimorphic yeast Schizosaccharomyces japonicus has also been used for genetic analyses of hyphal induction. We confirmed that Ras1-MAPK and other MAPK pathways exist in Sz. japonicus. To examine how hyphal transition is induced by environmental stress-triggered signal transduction, we studied the hyphal transition of deletion mutants of MAPK pathways in Sz. japonicus. We found that the MAPK pathways are not involved in hyphal induction, although the mating response is dependent on these pathways. However, only Ras1 deletion caused a severe defect in hyphal development via both DNA damage and environmental stressors. In fact, genes on the Cdc42 branch of the Ras1 (Ras1-Cdc42) pathway, efc25Sj, scd1Sj and scd2Sj, are required for hyphal development. Cell morphology analysis indicated that the apical growth of hyphal cells was inhibited in Ras1-Cdc42-pathway deletion mutants. Thus, the control of cell polarity by the Ras1-Cdc42 pathway is crucial for hyphal development.