An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation

Römermann, D; Warrelmann, Jürgen; Bender, R.; Friedrich, Bärbel

doi:10.1128/jb.171.2.1093-1099.1989

Cited by 62 publications

(42 citation statements)

References 41 publications

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“…Alternatively, P. syringae could have a single gene encoding glutamine synthetase, which requires 54 for the high-level expression needed for ammonia assimilation but which has sufficient basal expression to prevent auxotrophy. rpoN mutants of most soil bacteria, including Azotobacter vinelandii, P. putida, and Agrobacterium tumefaciens, are not glutamine auxotrophs (39,40,53,74,75).…”

Section: Discussionmentioning

confidence: 99%

Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

et al. 2000

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We cloned the rpoN (ntrA and glnF) gene encoding 54 from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the 54 protein encoded by the Pseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation, rpoN::Km r . In contrast to wild-type ES4326, ES4326 rpoN::Km r was nonmotile and could not utilize nitrate, urea, C 4 -dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326 rpoN::Km r did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Arabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Km r carrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326 rpoN::Km r partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of hrpL in ES4326 rpoN::Km r did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.The rpoN gene encodes the alternate sigma factor 54 , which works in conjunction with the NtrC class of transcriptional activators to control the expression of many genes in response to nutritional and environmental conditions (2, 54). For example, genes involved in nitrogen, hydrogen, and catabolite utilization are frequently regulated by 54 (6, 35, 48, 95). In the case of pathogenic bacteria, rpoN mediates expression of virulence-related factors such as pilin in Pseudomonas aeruginosa and flagellin in Vibrio anguillarum (24,61,86).For some phytopathogenic bacteria, rpoN has been implicated indirectly as a regulator of pathogenicity-related genes known as the hrp gene cluster (17, 18). Pseudomonas syringae pv. syringae strain 61, for example, contains a 25-kb hrp cluster consisting of several complementation groups comprising at least 27 genes (8, 26). Several hrp genes encode proteins that have a high degree of homology to components of the type III secretory pathway of Yersinia species which are responsible for translocating Yersinia outer membrane proteins into mammalian cells (13,15,55,83). By analogy, it is ...

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Section: Discussionmentioning

confidence: 99%

Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

et al. 2000

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“…The structural genes for the a-and P-subunits of acetoin:2,6-dichlorophenolindophenol oxidoreductase (acoA and acoB, respectively); for the protein FMP (acoC), which seems to be a dihydrolipoamide acetyltransferase; and for a protein of yet unknown function (acoX) are probably organized in one single operon (acoXABC) which is localized on fragments A and C (36). Fragment B harbors an rpoN-like gene, and mutants carrying a TnS insertion in this gene exhibited an extremely pleiotropic phenotype (13,38). As these mutants * Corresponding author.…”

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Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

et al. 1992

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The N-terminal amino acid sequence of purified acetaldehyde dehydrogenase II (AcDH-II) from ethanolgrown cells ofAlcaligenes eutrophus was determined. By using oligonucleotides deduced from this sequence the structural gene for AcDH-II, which was referred to as acoD, was localized on a 7.2-kbp EcoRI restriction fragment (fragment D), which has been cloned recently (C. Frund, H. Priefert, A. Steinbuchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989 The aerobic bacterium Alcaligenes eutrophus H16 synthesizes two different NAD-dependent acetaldehyde dehydrogenases (AcDH-I and AcDH-II; EC 1.2.1.3) during growth on acetoin. Both dehydrogenases are also synthesized in the type strain TF93 and in ethanol-utilizing mutants of A. eutrophus derived from strains H16 or N9A during growth on ethanol or on acetoin. Since the expression of AcDH-II is specifically induced only during growth on these substrates, this enzyme seems to be responsible for the oxidation of acetaldehyde to acetate in both catabolic pathways. AcDH-II possesses a high affinity toward acetaldehyde (Km = 4 ,uM), exhibits a wide substrate spectrum, and amounts to about 14% of the total soluble protein in ethanol-grown cells (21).In A. eutrophus the degradation of acetoin is initiated by direct cleavage into two C2 compounds (13). This reaction is catalyzed by acetoin:2,6-dichlorophenolindophenol oxidoreductase and occurs also in Bacillus subtilis (28) and in the anaerobic bacterium Pelobacter carbinolicus (33) during growth on acetoin. Recently, we have identified and localized the genes, which are essentially involved in the catabolism of acetoin in A. eutrophus, on five different genomic EcoRI restriction fragments (A, B, C, D, and E) (13). The structural genes for the a-and P-subunits of acetoin:2,6-dichlorophenolindophenol oxidoreductase (acoA and acoB, respectively); for the protein FMP (acoC), which seems to be a dihydrolipoamide acetyltransferase; and for a protein of yet unknown function (acoX) are probably organized in one single operon (acoXABC) which is localized on fragments A and C (36). Fragment B harbors an rpoN-like gene, and mutants carrying a TnS insertion in this gene exhibited an extremely pleiotropic phenotype (13,38). As these mutants * Corresponding author.were also unable to utilize acetoin as a sole carbon source for growth, the expression of acetoin-catabolic genes in A. eutrophus seems to be also rpoN dependent. This is consistent with the identification of a promoter exhibiting striking homology to the enterobacterial c54-dependent promoter consensus sequence upstream of acoX-ABC. The information encoded by fragments D and E remained to be elucidated.In the present study we localized the structural gene for AcDH-II (acoD), which is in A. eutrophus essentially involved in the catabolism of acetoin as well as of ethanol, on EcoRI restriction fragment D. The results of the molecular characterization of acoD and of adjacent DNA regions confirmed our assumption that the expression of acoD is also rpoN dependent, like the ex...

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“…Furthermore, the synthesis of both hydrogenases is temperature sensitive; hydrogenase synthesis is completely abolished above 33°C (10). The expression of both hydrogenase proteins depends on a chromosomal gene, designated hno, which controls a number of metabolic activities in A. eutrophus (34). Recent nucleotide sequence analysis indicated that hno encodes a54 of core RNA polymerase (50).…”

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Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus

Eberz

Friedrich

1991

J Bacteriol

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Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.

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An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation

Cited by 62 publications

References 41 publications

Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus

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