The alternative sigma factor σ28 of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant

Studholme, David J.

doi:10.1016/s0378-1097(00)00378-5

Cited by 6 publications

(6 citation statements)

References 16 publications

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“…Whereas individual alternate sigma factors are generally involved in expression of related functions, the σ 54 type are atypical in that they are involved in the initiation of transcription of a range of different cellular functions, including the transport and metabolism of diverse nitrogen and carbon sources, plant pathogenicity, polysaccharide biosynthesis, etc. (Studholme and Buck, 2000; Cases et al ., 2003). Like P. aeruginosa , the KT2440 genome specifies 22 σ 54 (RpoN)‐dependent transcriptional regulators, which are considerably more than those in the habitat‐related microorganisms M. loti (13) and S. meliloti (12), A. tumefaciens (9), R. solanacearum (8) and C. crescentus (5), and far more than most other prokaryotes sequenced to date.…”

Section: Resultsmentioning

confidence: 99%

Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440

Santos

Heim

Moore

et al. 2004

Environmental Microbiology

249

202

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A major challenge in microbiology is the elucidation of the genetic and ecophysiological basis of habitat specificity of microbes. Pseudomonas putida is a paradigm of a ubiquitous metabolically versatile soil bacterium. Strain KT2440, a safety strain that has become a laboratory workhorse worldwide, has been recently sequenced and its genome annotated. By drawing on both published information and on original in silico analysis of its genome, we address here the question of what genomic features of KT2440 could explain or are consistent with its ubiquity, metabolic versatility and adaptability. The genome of KT2440 exhibits combinations of features characteristic of terrestrial, rhizosphere and aquatic bacteria, which thrive in either copiotrophic or oligotrophic habitats, and suggests that P. putida has evolved and acquired functions that equip it to thrive in diverse, often inhospitable environments, either free-living, or in close association with plants. The high diversity of protein families encoded by its genome, the large number and variety of small aralogous families, insertion elements, repetitive extragenic palindromic sequences, as well as the mosaic structure of the genome (with many regions of 'atypical' composition) and the multiplicity of mobile elements, reflect a high functional diversity in P. putida and are indicative of its evolutionary trajectory and adaptation to the diverse habitats in which it thrives. The unusual wealth of determinants for high affinity nutrient acquisition systems, mono- and di-oxygenases, oxido-reductases, ferredoxins and cytochromes, dehydrogenases, sulfur metabolism proteins, for efflux pumps and glutathione-S-transfereases, and for the extensive array of extracytoplasmatic function sigma factors, regulators, and stress response systems, constitute the genomic basis for the exceptional nutritional versatility and opportunism of P. putida , its ubiquity in diverse soil, rhizosphere and aquatic systems, and its renowned tolerance of natural and anthropogenic stresses. This metabolic diversity is also the basis of the impressive evolutionary potential of KT2440, and its utility for the experimental design of novel pathways for the catabolism of organic, particularly aromatic, pollutants, and its potential for bioremediation of soils contaminated with such compounds as well as for its application in the production of high-added value compounds.

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Section: Resultsmentioning

confidence: 99%

Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440

Santos

Heim

Moore

et al. 2004

Environmental Microbiology

249

202

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“…Similar results were previously observed with A. aeolicus fliA ϩ from E. coli. (44). Interestingly, a P araB -C. trachomatis rspD ϩ plasmid did not restore motility in E. coli with a fliA deletion (39).…”

Section: A Aeolicus and C Trachomatismentioning

confidence: 99%

Genetic Transplantation: Salmonella enterica Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in GeneticallyIntractable Systems

Karlinsey

Hughes

2006

J Bacteriol

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In Salmonella enterica serovar Typhimurium, 28 and anti-sigma factor FlgM are regulatory proteins crucial for flagellar biogenesis and motility. In this study, we used S. enterica serovar Typhimurium as an in vivo heterologous system to study 28 and anti-28 interactions in organisms where genetic manipulation poses a significant challenge due to special growth requirements. The chromosomal copy of the S. enterica serovar Typhimurium 28 structural gene fliA was exchanged with homologs of Aquifex aeolicus (an extreme thermophile) and Chlamydia trachomatis (an obligate intracellular pathogen) by targeted replacement of a tetRA element in the fliA gene location using -Red-mediated recombination. The S. enterica serovar Typhimurium hybrid strains showed 28 -dependent gene expression, suggesting that 28 activities from diverse species are preserved in the heterologous host system. A. aeolicus mutants defective for 28 /FlgM interactions were also isolated in S. enterica serovar Typhimurium. These studies highlight a general strategy for analysis of protein function in species that are otherwise genetically intractable and a straightforward method of chromosome restructuring using -Red-mediated recombination.

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“…Concluding remarks. Recognition of flagellin promoters by D orthologs is conserved across distantly related species (3,11,27). We here demonstrate this conservation for A. pallidus; the hag promoter, like that in B. subtilis (6), is D dependent and appears to include a strong UP element.…”

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confidence: 99%

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins

et al. 2011

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D proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the ؊10 element, and this motif is known to be recognized by region 2.4 and critical for promoter activity.In the course of efforts to define gene expression determinants from the thermophilic bacterium Aeribacillus pallidus AC6 (2, 18), we identified flagellin (Hag) as being among the most highly expressed proteins in this strain. To determine the basis for high-level Hag expression, we isolated and sequenced the hag gene and identified and expressed the protein required for its expression in The resulting peptide sequences displayed high similarity to B. subtilis flagellin (NAQDGISLIQTAEGALTETHAILQR had 96% identity with amino acids [aa] 65 to 89 and LEHTINNL GTSAENLTAAESR had 85% identity with aa 242 to 262). Two degenerate primers (FlaF1 and FlaR1) were used to amplify the flagellin gene (hag) from A. pallidus AC6 chromosomal DNA. An ϳ450-bp product was cloned into pGEM-T Easy (Promega) for DNA sequencing. The remainder of hag and its upstream region were obtained by inverse PCR. The 828-bp hag gene encodes a 275-amino-acid (29.7-kDa) protein having 63% identity with B. subtilis Hag and is preceded by a typical D promoter. To identify sigD, two degenerate primers were used to amplify a PCR fragment which was cloned into the pGEM-T Easy vector system and sequenced. The flanking portions of sigD were obtained by inverse PCR, and the gene was sequenced. The 771-bp sigD gene encodes a 256-amino-acid (28.7-kDa) protein having 67% overall identity with D Bs , with the highest levels of similarity concentrated in conserved regions 2 and 4, known to mediate promoter recognition.A. pallidus AC6 flagellin is expressed from a D -dependent promoter. Transcription of hag initiates from a canonical Ddependent promoter at a G residue 79 bp upstream of the start codon (Fig. 1). Analysis of hag::lacZ fusions integrated into B. subtilis CU1065 and HB4035 (sigD::kan) indicated that activity was D dependent and highest at late logarithmic phase, as previously reported for B. subtilis (19). Optimal promoter activity required an AT-rich region just upstream of the Ϫ35 element (Table 1), which has similarity with the upstream promoter (UP) element previously described for B. subtilis hag (6). Sequence inspection suggests that high-level Hag expression may also benefit from a strong ribosome-binding site and stabilization of the mRNA by a 5Ј hairpin sequence (24). Purification D of A. pallidus and B. subtilis and reconstitution of D RNAP. D proteins from A. pallidus AC6 and B. subtilis were expressed under T7 RNA polymerase (RNAP) control in Escherichia coli BL21(DE3)/pLysS (Novagen) by using pECG1 and pECB1 (Table 2). For purification, inclusion bodies were solubilized with Sarkosyl (1), refolded, and purified using DEAE-Sepharose and heparin-Sepharose chromatography as described previously (9). B. subtilis core RNAP was purified...

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The alternative sigma factor σ28 of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant

Cited by 6 publications

References 16 publications

Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440

Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440

Genetic Transplantation: Salmonella enterica Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in GeneticallyIntractable Systems

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins

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The alternative sigma factor σ28 of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant

Cited by 6 publications

References 16 publications

Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440

Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440

Genetic Transplantation: Salmonella enterica Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in GeneticallyIntractable Systems

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ D Proteins

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DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ ^D Proteins