Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in <i>Myxococcus xanthus</i>

McCurdy, Howard D.; Ho, Joseph; Dobson, William J.

doi:10.1139/m78-237

Cited by 16 publications

(11 citation statements)

References 5 publications

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“…2). The result agrees with that of a previous study in which when M. xanthus inoculated on fruiting medium was treated with cNMP phosphodiesterase, fruiting body formation was accelerated compared with that of controls without cNMP phosphodiesterase (21). On the other hand, 5Ј-AMP .....................................................................................15.2 Ϯ 1.3 3Ј,5Ј-cGMP...............................................................................20.7 Ϯ 0.9 5Ј-GTP ...................................................................................... 5.0 Ϯ 0 5Ј-GDP ..................................................................................... 9.3 Ϯ 0.4 5Ј-GMP................................................................................ …”

supporting

confidence: 92%

Enzymatic and Mutational Analyses of a Class II 3',5'-Cyclic Nucleotide Phosphodiesterase, PdeE, from Myxococcus xanthus

Kimura¹,

Yoshimi²,

Takata³

2011

Journal of Bacteriology

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Myxococcus xanthus PdeE, an enzyme homologous to class II 3,5-cyclic nucleotide phosphodiesterases, hydrolyzed cyclic AMP (cAMP) and cGMP with K m values of 12 M and 25 M, respectively. A pdeE mutant exhibited delays in fruiting body and spore formation compared with the wild type when cultured on starvation medium.The second messengers cyclic AMP (cAMP) and cGMP are important regulators of numerous biological functions in microorganisms, such as the control of metabolic pathways in eubacteria, differentiation and virulence in fungi, and cell aggregation in Dictyostelium (3, 6). cAMP and cGMP are generated from ATP and GTP by adenylyl cyclase and guanylyl cyclase, respectively, and are degraded through phosphodiesterase hydrolysis. Myxococcus xanthus is a Gram-negative bacterium which demonstrates complex social behavior (11,27). M. xanthus has two receptor-type adenylyl cyclases, CyaA and CyaB, which are required for osmotic tolerance during spore germination and growth, respectively (12, 13). In addition, M. xanthus has at least two phosphodiesterases, PdeA and PdeB, of the class III type, which are confined to the prokaryotes; pdeA and pdeB mutants showed impaired growth under conditions of high-temperature stress (14). The spores of mutants placed under osmotic stress germinated earlier than wild-type spores. PdeA and PdeB hydrolyze 3Ј,5Ј-and 2Ј,3Ј-cAMP to adenosine and also demonstrate phosphatase activity toward nucleoside 5Ј-tri-, 5Ј-di-, 5Ј-and 3Ј-monophosphates, with highest activities for nucleoside 5Ј-monophosphates (NMP) (15). Class II phosphodiesterases are found in some prokaryotes (e.g., Vibrio fischeri) and in many lower eukaryotes, such as Dictyostelium discoideum or fungi, including Saccharomyces and Neurospora (2,5,18,28). Since there have been extremely few reports on the class II cNMP phosphodiesterases of bacteria, we analyzed the enzymatic properties and function of the class II-type cNMP phosphodiesterase PdeE of M. xanthus.Comparison of amino acid sequences of PdeE and other class II phosphodiesterases. The open reading frame of MXAN_2703, designated pdeE, encodes a protein of 251 amino acids with a calculated molecular mass of 27.6 kDa. The deduced PdeE amino acid sequence has the highest identity with those from the order Myxococcales, such as Stigmatella aurantiaca phosphodiesterase (80% identity) and Sorangium cellulosum phosphodiesterase (44% identity) (Fig. 1). They contain the conserved sequence motif HXHXDH, where X is any residue, found in class II 3Ј,5Ј-cAMP phosphodiesterases (25). The deduced amino acid sequence of PdeE also showed 30% identity and 27% identity with that of the class II phosphodiesterase CpdP of V. fischeri (7) and that of the class II phosphodiesterase DdPDE1 of D. discoideum (16), respectively (data not shown).Expression of PdeE in Escherichia coli and cNMP hydrolysis. The pdeE gene amplified by PCR using the PdeEN and PdeEC primers (Table 1) was cloned into the NdeI/EcoRI sites of a His 6 -tagged expression vector pCold I (Takara Bio), and then the recombi...

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supporting

confidence: 92%

Enzymatic and Mutational Analyses of a Class II 3',5'-Cyclic Nucleotide Phosphodiesterase, PdeE, from Myxococcus xanthus

Kimura¹,

Yoshimi²,

Takata³

2011

Journal of Bacteriology

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“…Gliding Myxococcus cells deposit a slime trail, and the dye Congo red is adsorbed by Myxococcus colonies (29,30). Arnold and Shimkets (2) have correlated Congo red binding with a loose network of thick fibers that surrounds cells and adheres to agar.…”

Section: Resultsmentioning

confidence: 99%

“…Colonies were stained for slime components by flooding the colony with 400 ,ul of an aqueous solution of 0.02% Congo red and 100 mM sodium phosphate (pH 7.7) (2,29,30 (wild type), and DK1250 (A-S-) were grown in CTT broth to a cell density of 4 x 10' to 5 x 108 cell per ml (100 Klett units [20]) and adjusted to 5 and 25 Klett units by dilution in a 1:1 (by volume) CTT deionized water solution. Activated charcoal was added to each sample to a final concentration of 50 ,ug/ml.…”

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confidence: 99%

Upstream gene of the mgl operon controls the level of MglA protein in Myxococcus xanthus

Hartzell

Kaiser

1991

J Bacteriol

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The mgl operon contains two open reading frames (ORFs) which are transcribed together. A collection of nonmotile mutants helped to define the downstream ORF as the mglA gene. Single mutations at the mglA locus completely abolish motility. A series of deletion mutations was constructed to determine the role of the upstream ORF (now called mglB). A strain carrying a deletion in mglB and with an intact mglA produces small colonies. The cells are motile, but their rate of swarm spreading is reduced. Measurements of cell movement showed that mglB mutant cells advanced, on average, less than 0.1 cell length in 5 min. The mglB+ cells advanced an average of 1.3 cell lengths in the same time. Extracts of AmglB cells contain 15 to 20% as much of the 22-kDa MglA protein as do mglB+ cells, as measured in Western immunoblots and enzyme-linked immunosorbent assays. However, the amount of mgl transcript is the same in the AmglB mutants as in the mglB+ strain. Heterozygous partial diploids mglBImglA with the wild-type alleles in trans have normal motility, demonstrating that the largest of the mglB deletions is not polar on mglA. Like other motility defects, a AmglB mutation alters fruiting body development and sporulation. The mglB mutants delayed aggregation, produced small immature fruiting bodies, and sporulated at 45 to 50% wild-type levels. All aspects of the mglB mutant phenotype are explained by the reduced levels of mglA protein and the assumption that it limits the amount of gliding.Gliding motility is defined as translocation over a solid surface and involves movement in the direction of the long axis of the cell (8). In all known gliding organisms, movement is correlated with the production of slime. There is occasional reversal of direction, but motion is smooth, with no rotation apparent. No appendages, which would suggest a mechanism for gliding, have been identified. Gliding is employed by a diverse group of organisms, including Cytophaga and Flexibacter spp., the myxobacteria, and the filamentous cyanobacteria, all of which belong to evolutionarily different groups (33). These organisms range in length from 5 to 50 ,um and glide at rates of 1 to 300 pum/min. While different explanations for gliding have been proposed for different organisms (directional extrusion of slime [24], polar release of surfactant [12], and movement by contraction [8,26], for example), the molecular mechanism of gliding remains unsettled.Genes that regulate the pattern of gliding motility have been found. Two genetically independent sets of genes, called the adventurous (A) and social (S) motility systems, have been identified in Myxococcus xanthus (18,19

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“…Ho and McCurdy reported that the cellular concentration of cAMP in M. xanthus during development increased rapidly about 20-fold during the first 15 h of incubation and then decreased 75% by 25 h, and the rapid drop in cAMP concentration corresponds to an increase in cAMP phosphodiesterase activity which exhibited a maximum at 40 h (5). McCurdy et al also reported that exogenous cyclic nucleotide phosphodiesterase accelerates fruiting body formation in M. xanthus (16). These results suggest that cyclic nucleotide phosphodiesterases may be involved in the development of M. xanthus, however, ablation of the single cyclic nucleotide phosphodiesterase gene (pdeA, pdeB, or pdeC) has no noticeable effect on development.…”

Section: Fig 2 Amino Acid Sequence Alignment Of Homologous Regions mentioning

confidence: 99%

Contribution of the Cyclic Nucleotide Phosphodiesterases PdeA and PdeB to Adaptation of Myxococcus xanthus Cells to Osmotic or High-Temperature Stress

et al. 2006

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A tBLASTn search of the Myxococcus xanthus genome database at The Institute for Genomic Research (TIGR) identified three genes (pdeA, pdeB, and pdeC) that encode proteins homologous to 3,5-cyclic nucleotide phosphodiesterase. pdeA, pdeB, and pdeC mutants, constructed by replacing a part of the gene with the kanamycin or tetracycline resistance gene, showed normal growth, development, and germination under nonstress conditions. However, the spores of mutants, especially the pdeA and pdeB mutants, placed under osmotic stress germinated earlier than the wild-type spores. The phenotype was the opposite of that of the receptor-type adenylyl cyclase (cyaA or cyaB) mutant. Also, pdeA and pdeB mutants were found to have impaired growth under the condition of high-temperature stress. Intracellular cyclic AMP (cAMP) levels of pdeA or pdeB mutant cells under these stressful conditions were about 1.3-fold to 2.0-fold higher than those of wild-type cells. These results suggest that PdeA and PdeB may be involved in osmotic adaptation during spore germination and temperature adaptation during vegetative growth through the regulation of cAMP levels.

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Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in Myxococcus xanthus

Cited by 16 publications

References 5 publications

Enzymatic and Mutational Analyses of a Class II 3',5'-Cyclic Nucleotide Phosphodiesterase, PdeE, from Myxococcus xanthus

Enzymatic and Mutational Analyses of a Class II 3',5'-Cyclic Nucleotide Phosphodiesterase, PdeE, from Myxococcus xanthus

Upstream gene of the mgl operon controls the level of MglA protein in Myxococcus xanthus

Contribution of the Cyclic Nucleotide Phosphodiesterases PdeA and PdeB to Adaptation of Myxococcus xanthus Cells to Osmotic or High-Temperature Stress

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