scholarly journals pbpB, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium Anabaena sp. Strain PCC7120

2001 ◽  
Vol 183 (2) ◽  
pp. 628-636 ◽  
Author(s):  
Sara Lázaro ◽  
Francisca Fernández-Piñas ◽  
Eduardo Fernández-Valiente ◽  
Amaya Blanco-Rivero ◽  
Francisco Leganés
Keyword(s):  
Sequence Similarity ◽  
Shuttle Vector ◽  
Molecular Size ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Reading Frame ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Peptidoglycan Layer

ABSTRACT Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequencedAnabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and theEscherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal aspbpB and its product as PBP2. The wild-type form ofpbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N2, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

mrpA, a gene with roles in resistance to Na+ and adaptation to alkaline pH in the cyanobacterium Anabaena sp. PCC7120

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1671-1682 ◽  
Author(s):  
A. Blanco-Rivero ◽  
F. Leganés ◽  
E. Fernández-Valiente ◽  
P. Calle ◽  
F. Fernández-Piñas
Keyword(s):  
Mutant Strain ◽  
Sequence Similarity ◽  
Biological Significance ◽  
Proton Pumping ◽  
Alkaline Ph ◽  
Wild Type ◽  
Significant Similarity ◽  
Cyanobacterium Anabaena ◽  
In The Wild

Transposon mutagenesis of Anabaena sp. PCC7120 led to the isolation of a mutant strain, PHB11, which grew poorly at pH values above 10. The mutant strain exhibited pronounced Na+ sensitivity; this sensitivity was higher under basic conditions. Mutant PHB11 also showed an inhibition of photosynthesis that was much more pronounced at alkaline pH. Reconstruction of the transposon mutation of PHB11 in the wild-type strain reproduced the phenotype of the original mutant. The wild-type version of the mutated gene was cloned and the mutation complemented. In mutant strain PHB11, the transposon had inserted within an ORF that is part of a seven-ORF operon with significant sequence similarity to a family of bacterial operons that are believed to code for a novel multiprotein cation/proton antiporter primarily involved in resistance to salt stress and adaptation to alkaline pH. The Anabaena operon was denoted mrp (multiple resistance and pH adaptation) following the nomenclature of the Bacillus subtilis operon; the ORF mutated in PHB11 corresponded to mrpA. Computer analysis suggested that all seven predicted Anabaena Mrp proteins were highly hydrophobic with several transmembrane domains; in fact, the predicted protein sequences encoded by mrpA, mrpB and mrpC showed significant similarity to hydrophobic subunits of the proton pumping NADH : ubiquinone oxidoreductase. In vivo expression studies indicated that mrpA is induced with increasing external Na+ concentrations and alkaline pH; mrpA is also upregulated under inorganic carbon (Ci) limitation. The biological significance of a putative cyanobacterial Mrp complex is discussed.

scholarly journals Heterocyst Development and Diazotrophic Metabolism in Terminal Respiratory Oxidase Mutants of the Cyanobacterium Anabaena sp. Strain PCC 7120

2007 ◽  
Vol 189 (12) ◽  
pp. 4425-4430 ◽  
Author(s):  
Ana Valladares ◽  
Iris Maldener ◽  
Alicia M. Muro-Pastor ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Nitrogenase Activity ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Membrane Reorganization ◽  
Respiratory Oxidases ◽  
And Performance ◽  
Pcc 7120 ◽  
Heterocyst Development

ABSTRACT Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N2 fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.

scholarly journals HcwA, an Autolysin, Is Required for Heterocyst Maturation in Anabaena sp. Strain PCC 7120

2001 ◽  
Vol 183 (23) ◽  
pp. 6841-6851 ◽  
Author(s):  
Jinsong Zhu ◽  
Karin Jäger ◽  
Todd Black ◽  
Kelly Zarka ◽  
Olga Koksharova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Open Reading Frame ◽  
Mutant Phenotype ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Filamentous Cyanobacteria ◽  
Reading Frame ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Peptidoglycan Layer

ABSTRACT In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N2. Synthesis of the heterocyst envelope polysaccharide is dependent on the gene hepA inAnabaena sp. strain PCC 7120. In search of genes that are involved in the regulation of hepA, we transposon mutagenized strain DR1069, which bears a chromosomalhepA::luxAB fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of hepA in response to nitrogen deprivation. In HNL3, transposon Tn5-1058 is inserted within gene hcwA, a constitutively expressed open reading frame whose predicted product resemblesN-acetylmuramoyl-l-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction ofhepA in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type hcwA. Moreover, HcwA expressed in Escherichia coli exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

scholarly journals MyD88-Dependent Signaling Affects the Development of Meningococcal Sepsis by Nonlipooligosaccharide Ligands

2006 ◽  
Vol 74 (6) ◽  
pp. 3538-3546 ◽  
Author(s):  
Laura Plant ◽  
Hong Wan ◽  
Ann-Beth Jonsson
Keyword(s):  
Inflammatory Responses ◽  
Innate Immune ◽  
Meningococcal Sepsis ◽  
Wild Type ◽  
Microbial Infections ◽  
In The Wild ◽  
Myeloid Differentiation Factor ◽  
Dependent Pathway ◽  
Meningococcal Strain

ABSTRACT The Toll-like receptors (TLRs) and the adaptor myeloid differentiation factor 88 (MyD88) are important in the innate immune defenses of the host to microbial infections. Meningococcal ligands signaling via TLRs control inflammatory responses, and stimulation can result in fulminant meningococcal sepsis. In this study, we show that the responses to nonlipooligosaccharide (non-LOS) ligands of meningococci are MyD88 dependent. An isogenic LOS-deficient mutant of the serogroup C meningococcal strain FAM20 caused fatal disease in wild type C57BL/6 mice that was not observed in MyD88−/− mice. Fatality correlated with high proinflammatory cytokine and C5a levels in serum, high neutrophil numbers in blood, and increased bacteremia at 24 h postinfection in the wild-type mice. Infection with the parent strain FAM20 resulted in fatality in 100% of the wild-type mice and 50% of the MyD88−/− mice. We conclude that both LOS and another neisserial ligand cause meningococcal sepsis in an in vivo mouse model and confirm that meningococcal LOS can act via both the MyD88- dependent and -independent pathways, while the non-LOS meningococcal ligand(s) acts only via the MyD88-dependent pathway.

Effect offurmutation on acid-tolerance response and in vivo virulence of avian septicemicEscherichia coli

2002 ◽  
Vol 48 (5) ◽  
pp. 458-462 ◽  
Author(s):  
Chengru Zhu ◽  
Musangu Ngeleka ◽  
Andrew A Potter ◽  
Brenda J Allan
Keyword(s):  
Parent Strain ◽  
Acid Tolerance ◽  
Wild Type ◽  
Acid Tolerance Response ◽  
E Coli ◽  
Regulator Protein ◽  
In The Wild ◽  
Tolerance Response ◽  
Wild Type Parent

The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria. In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined. The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain. However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation. These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E. coli, as detected by the ability to cause septicemia in our experimental infection.Key words: E. coli, fur, acid-tolerance response.

scholarly journals The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

1998 ◽  
Vol 18 (3) ◽  
pp. 1506-1516 ◽  
Author(s):  
Ying Cui ◽  
Jonathan D. Dinman ◽  
Terri Goss Kinzy ◽  
Stuart W. Peltz
Keyword(s):  
Site Selection ◽  
Start Site ◽  
Wild Type ◽  
Virus Propagation ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Translational Accuracy ◽  
Its Gene ◽  
Killer Virus

ABSTRACT Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene.mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

scholarly journals Replication Properties of Clade A/C Chimeric Feline Immunodeficiency Viruses and Evaluation of Infection Kinetics in the Domestic Cat

2008 ◽  
Vol 82 (16) ◽  
pp. 7953-7963 ◽  
Author(s):  
Sohela de Rozìeres ◽  
Jesse Thompson ◽  
Magnus Sundstrom ◽  
Julia Gruber ◽  
Debora S. Stump ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Domestic Cat ◽  
Fine Tuning ◽  
Surface Glycoprotein ◽  
Wild Type ◽  
Coding Region ◽  
Mild Disease ◽  
Clade C ◽  
In The Wild

ABSTRACT Feline immunodeficiency virus (FIV) causes progressive immunodeficiency in domestic cats, with clinical course dependent on virus strain. For example, clade A FIV-PPR is predominantly neurotropic and causes a mild disease in the periphery, whereas clade C FIV-C36 causes fulminant disease with CD4+ T-cell depletion and neutropenia but no significant pathology in the central nervous system. In order to map pathogenic determinants, chimeric viruses were prepared between FIV-C36 and FIV-PPR, with reciprocal exchanges involving (i) the 3′ halves of the viruses, including the Vif, OrfA, and Env genes; (ii) the 5′ end extending from the 5′ long terminal repeat (LTR) to the beginning of the capsid (CA)-coding region; and (iii) the 3′ LTR and Rev2-coding regions. Ex vivo replication rates and in vivo replication and pathologies were then assessed and compared to those of the parental viruses. The results show that FIV-C36 replicates ex vivo and in vivo to levels approximately 20-fold greater than those of FIV-PPR. None of the chimeric FIVs recapitulated the replication rate of FIV-C36, although most replicated to levels similar to those of FIV-PPR. The rates of chloramphenicol acetyltransferase gene transcription driven by the FIV-C36 and FIV-PPR LTRs were identical. Furthermore, the ratios of surface glycoprotein (SU) to capsid protein (CA) in the released particles were essentially the same in the wild-type and chimeric FIVs. Tests were performed in vivo on the wild-type FIVs and chimeras carrying the 3′ half of FIV-C36 or the 3′ LTR and Rev2 regions of FIV-C36 on the PPR background. Both chimeras were infectious in vivo, although replication levels were lower than for the parental viruses. The chimera carrying the 3′ half of FIV-C36 demonstrated an intermediate disease course with a delayed peak viral load but ultimately resulted in significant reductions in neutrophil and CD4+ T cells, suggesting potential adaptation in vivo. Taken together, the findings suggest that the rapid-growth phenotype and pathogenicity of FIV-C36 are the result of evolutionary fine tuning throughout the viral genome, rather than being properties of any one constituent.

scholarly journals Altered RNA Editing in Mice Lacking ADAR2 Autoregulation

2006 ◽  
Vol 26 (2) ◽  
pp. 480-488 ◽  
Author(s):  
Yi Feng ◽  
Christopher L. Sansam ◽  
Minati Singh ◽  
Ronald B. Emeson
Keyword(s):  
Alternative Splicing ◽  
Protein Expression ◽  
Translation Termination ◽  
Editing Site ◽  
Wild Type ◽  
Complementary Sequence ◽  
Double Stranded Rna ◽  
Reading Frame ◽  
Site Selective

ABSTRACT ADAR2 is a double-stranded-RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-selective conversion of adenosine to inosine. Previous studies from our laboratory have demonstrated that ADAR2 can modify its own pre-mRNA to create a proximal 3′ splice site containing a noncanonical adenosine-inosine dinucleotide. Alternative splicing to this proximal acceptor adds 47 nucleotides to the mature ADAR2 transcript, thereby resulting in the loss of functional ADAR2 protein expression due to premature translation termination in an alternate reading frame. To examine whether the editing of ADAR2 transcripts represents a negative autoregulatory strategy to modulate ADAR2 protein expression, we have generated genetically modified mice in which the ability of ADAR2 to edit its own pre-mRNA has been selectively ablated by deletion of a critical sequence (editing site complementary sequence [ECS]) required for adenosine-to-inosine conversion. Here we demonstrate that ADAR2 autoediting and subsequent alternative splicing are abolished in homozygous ΔECS mice and that ADAR2 protein expression is increased in numerous tissues compared to wild-type animals. The observed increases in ADAR2 protein expression correlate with the extent of ADAR2 autoediting observed with wild-type tissues and correspond to increases in the editing of ADAR2 substrates, indicating that ADAR2 autoediting is a key regulator of ADAR2 protein expression and activity in vivo.

scholarly journals Pyruvate Formate Lyase Is Required for Pneumococcal Fermentative Metabolism and Virulence

2009 ◽  
Vol 77 (12) ◽  
pp. 5418-5427 ◽  
Author(s):  
Hasan Yesilkaya ◽  
Francesca Spissu ◽  
Sandra M. Carvalho ◽  
Vanessa S. Terra ◽  
Karen A. Homer ◽  
...  
Keyword(s):  
Wild Type ◽  
Product Analysis ◽  
Acid Fermentation ◽  
Pyruvate Formate Lyase ◽  
Growth And Survival ◽  
Mixed Acid Fermentation ◽  
Mixed Acid ◽  
Fermentative Metabolism ◽  
In The Wild

ABSTRACT Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pfl A genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.

Sign in / Sign up

Export Citation Format

Share Document