Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.     

Characterization of devA, a gene required for the maturation of proheterocysts in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant M7, obtained by transposon mutagenesis of the cyanobacterium Anabaena sp. strain PCC 7120, is impaired in the development of mature heterocysts. Under aerobic conditions, the mutant is unable to fix N2 because of a deficiency of at least two components of the oxygen-protective mechanisms: a hemoprotein-coupled oxidative reaction and heterocyst-specific glycolipids. DNA contiguous with the inserted transposon was recovered from the mutant and sequenced. The transposon had inserted itself within a 732-bp open reading frame designated devA. The wild-type form of devA, obtained from a lambda-EMBL3 library of Anabaena sp. DNA, had the identical sequence. Directed mutagenesis of devA in the wild-type strain showed that the phenotype of the mutant was caused by insertion of the transposon. The wild-type form of devA on a shuttle vector complemented the mutation in M7. Expression of devA by whole filaments, monitored following nitrogen stepdown by using luxAB as the reporter, increased ca. eightfold during differentiation; the increase within differentiating cells was much greater. The deduced sequence of the DevA protein shows strong similarity to the ATP-binding subunit of binding protein-dependent transport systems. The product of devA may, therefore, be a component of a periplasmic permease that is required for the transition from a proheterocyst to a mature, nitrogen-fixing heterocyst.     

Tunicamycin suppresses differentiation and stimulates dedifferentiation of heterocysts in the cyanobacteriumAnabaena azollae Stras

The effect of tunicamycin (TM), a specific inhibitor of glycosylation of proteins, on heterocyst differentiation inAnabaena azollae Stras. was investigated. Heterocysts were developed in the presence of TM up to a concentration of 0.2 g ml^–1, whereas at higher concentrations differentiation proceeded only up to the proheterocyst stage. Analysis of lipids by thinlayer chromatography showed that the glycolipid that is specific for the laminated layer of mature heterocysts was synthesized even in the cultures where the differentiation had proceeded only up to the early proheterocyst stage (i.e., at>0.2 g TM ml^–1). Further, deposition of the glycolipid-containing laminated layer in the envelope of the heterocysts differentiated in the presence of TM (i.e., at 0.2 g ml^–1) was incohesive as observed at the ultrastructural level. These findings clearly suggest that the process leading to the transportation of the heterocyst-specific glycolipid and its deposition as a laminated layer are affected by TM treatment. Because of the reported highly selective mode of action of TM, our results implicate a role for protein glycosylation in the process of heterocyst development and maturation.Although the heterocyst is a terminal stage of differentiation, because they normally do not divide/multiply, a considerable percentage of heterocysts that developed in the presence of TM dedifferentiated. The aberrations formed in the laminated layer, probably because of the inhibition of protein glycosylation owing to TM treatment, may have contributed to the instability of heterocyst structure and consequently led to their dedifferentiation.     

PrpJ, a PP2C-type protein phosphatase located on the plasma membrane, is involved in heterocyst maturation in the cyanobacterium Anabaena sp. PCC 7120

Protein phosphatases play important roles in the regulation of cell growth, division and differentiation. The cyanobacterium Anabaena PCC 7120 is able to differentiate heterocysts specialized in nitrogen fixation. To protect the nitrogenase from inactivation by oxygen, heterocyst envelope possesses a layer of polysaccharide and a layer of glycolipids. In the present study, we characterized All1731 (PrpJ), a protein phosphatase from Anabaena PCC 7120. prpJ was constitutively expressed in both vegetative cells and heterocysts. Under diazotrophic conditions, the mutant DeltaprpJ (S20) did not grow, lacked only one of the two heterocyst glycolipids, and fragmented extensively at the junctions between developing cells and vegetative cells. No heterocyst glycolipid layer could be observed in the mutant by electron microscopy. The inactivation of prpJ affected the expression of hglE(A) and nifH, two genes necessary for the formation of the glycolipid layer of heterocysts and the nitrogenase respectively. PrpJ displayed a phosphatase activity characteristic of PP2C-type protein phosphatases, and was localized on the plasma membrane. The function of prpJ establishes a new control point for heterocyst maturation because it regulates the synthesis of only one of the two heterocyst glycolipids while all other genes so far analysed regulate the synthesis of both heterocyst glycolipids.     

Lipids of membranes and of the cell envelope in heterocysts of a blue-green alga

Summary Heterocysts of Anabaena cylindrica, isolated rapidly in the cold, were found—in contrast to earlier reports—to contain all of the same lipids and lipophilic pigments, and in about the same proportions, as vegetative cells. In broken filaments and in heterocysts damaged during isolation, the membrane lipids and certain pigments (myxoxanthophyll and an unidentified red pigment) break down rapidly. The glycolipids specific to heterocyst-forming blue-green algae are localized in the laminated layer of the heterocyst envelope. A possible role of the laminated layer is discussed.     

Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

A TolC-like protein is required for heterocyst development in Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane beta-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).     

The hglK gene is required for localization of heterocyst-specific glycolipids in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant strain 543 of the cyanobacterium Anabaena sp. strain PCC 7120 was originally isolated as a Fox- mutant following chemical mutagenesis. Ultrastructural analysis shows that in nitrogen-replete media the vegetative cells of the mutant are more cylindrical and have thicker septa than those of the wild type, while in nitrogen-free media the mutant heterocysts lack the normal glycolipid layer external to the cell wall. Although this layer is absent, strain 543 heterocysts nevertheless contain heterocyst-specific glycolipids, as determined by thin-layer chromatography. The mutation in strain 543 is in a gene we have named hglK, encoding a protein of 727 amino acids. The wild-type HglK protein appears to contain four membrane-spanning regions followed by 36 repeats of a degenerate pentapeptide sequence, AXLXX. The mutation in strain 543 introduces a termination codon immediately upstream of the pentapeptide repeat region. A mutant constructed by insertion of an antibiotic resistance cassette near the beginning of the hglK gene has the same phenotype as strain 543. We propose that hglK encodes a protein necessary for the localization of heterocyst glycolipids and that this function requires the pentapeptide repeats of the HglK protein.     

Expression of the 49 human ATP binding cassette (ABC) genes in pluripotent embryonic stem cells and in early- and late-stage multipotent mesenchymal stem cells

The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporter^low phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.     

Predicted glycosyl transferase genes located outside the HEP island are required for formation of heterocyst envelope polysaccharide in Anabaena sp. strain PCC 7120

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.     

Caenorhabditis elegans ABCRNAi transporters interact genetically with rde-2 and mut-7

RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABC(RNAi) mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABC(RNAi) gene class. Genetic complementation tests reveal functions for ABC(RNAi) transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABC(RNAi) proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABC(RNAi) mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABC(RNAi) gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABC(RNAi) transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity.     

Boron Requirement for Envelope Structure and Function in Anabaena PCC 7119 Heterocysts

The effects of boron deficiency on the ultrastructure and envelopecomposition of heterocysts in the filamentous cyanobacteriumAnabaena PCC 7119 were studied. Microscopic examination of boron-deficientcultures showed changes in heterocyst morphology. When thesecells were isolated and their glycolipid content determined,this specific component of the laminated layer of the heterocystenvelope was found to be lacking. The evidence presented supportsthe view that boron plays an essential role in the structureand function of the heterocyst envelope. Key words: Anabaena, boron, heterocysts, nitrogenase, oxygen-protection     

Clustered genes required for synthesis and deposition of envelope glycolipids in Anabaena sp. strain PCC 7120

Photoreduction of dinitrogen by heterocyst-forming cyanobacteria is of great importance ecologically and for subsistence rice agriculture. Their heterocysts must have a glycolipid envelope layer that limits the entry of oxygen if nitrogenase is to remain active to fix dinitrogen in an oxygen-containing milieu (the Fox+ phenotype). Genes alr5354 (hglD), alr5355 (hglC) and alr5357 (hglB) of the filamentous cyanobacterium, Anabaena sp. strain PCC 7120, and hglE of Nostoc punctiforme are required for synthesis of heterocyst envelope glycolipids. Newly identified Fox- mutants bear transposons in nearby open reading frames (orfs) all5343, all5345-asr5349 and alr5351-alr5358. Complementation and other analysis provide evidence that at least orfs all5343 (or a co-transcribed gene), all5345, all5347, alr5348, asr5350-alr5353 and alr5356, but not asr5349, are also required for a Fox+ phenotype. Lipid and sequence analyses suggest that alr5351-alr5357 encode the enzymes that biosynthesize the glycolipid aglycones. Electron microscopy indicates a role of all5345 through all5347 in the normal deposition of the envelope glycolipids.