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The nucleotide sequence and location of the genes that mediate the synthesis of catechol from aniline was analyzed in the Pseudomonas putida UCC22 mt-2 chromosome. Introduction of the plasmid pTDN1-1016 into Pseudomonas putida UCC22 enabled the host to grow or metabolize methylated amines such as aniline and m-toluidine. Nucleotide sequence of the 9,233-bp HindIII (HC) fragment of pTDN1 indicated the presence of protein-encoding open reading frames which were designated as tnpA. Furthermore, the gene cluster in the HC fragment encoded large and small subunits of a terminal dioxygenase.

The 2,4-dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate dioxygenase (TfdA) of Alcaligenes eutrophus was analyzed by spectrophotometric assay with 4-nitrophenoxyacetic acid (4-NPAA). Spectrophometric analysis of the 2,4D-catalyzing enzymes of Alcaligenes eutrophus produced negative results for 4-NPAA degradation and exhibited no activity in the extracts. Furthermore, the TfdA isolates from Alcaligenes eutrophus did not exhibit color production in selected whole cells due to the inability of these cultures to transport 4-NPAA.

Summary: The gene context in microorganism genomes is of considerable help for identifying potential substrates. The C785_RS13685 gene in Herbaspirillum huttiense IAM 15032 is a member of the d‐altronate dehydratase protein family, and which functions as a d‐arabinonate dehydratase in vitro, is clustered with genes related to putative pentose metabolism. In the present study, further biochemical characterization and gene expression analyses revealed that l‐xylonate is a physiological substrate that is ultimately converted to α‐ketoglutarate via so‐called Route II of a non‐phosphorylative pathway. Several hexonates, including d‐altronate, d‐idonate and l‐gluconate, which are also substrates of C785_RS13685, also significantly up‐regulated the gene cluster containing C785_RS13685, suggesting a possibility that pyruvate and d‐ or l‐glycerate were ultimately produced (novel Route III). On the contrary, ACAV_RS08155 of Acidovorax avenae ATCC 19860, a homologous gene to C785_RS13685, functioned as a d‐altronate dehydratase in a novel l‐galactose pathway, through which l‐galactonate was epimerized at the C5 position by the sequential activity of two dehydrogenases, resulting in d‐altronate. Furthermore, this pathway completely overlapped with Route III of the non‐phosphorylative l‐fucose pathway. The 'substrate promiscuity' of d‐altronate dehydratase protein(s) is significantly expanded to 'metabolic promiscuity' in the d‐arabinose, sugar acid, l‐fucose and l‐galactose pathways. [ABSTRACT FROM AUTHOR]

Summary Heat shock gene expression is regulated by the cellular level and activity of the stress sigma factor σ32 in Gram-negative bacteria. A toluene-resistant, temperature-sensitive derivative strain of Pseudomonas putida KT2442, designated KT2442-R2 (R2), accumulated several heat shock proteins (HSPs) under non-stress conditions. Genome sequencing of strain R2 revealed that its genome contains a number of point mutations, including a CGT to CCT change in dnaK resulting in an Arg445 to Pro substitution in DnaK. DNA microarray and real-time reverse transcription polymerase chain reaction analyses revealed that the mRNA levels of representative hsp genes (e.g. dnaK, htpG and groEL) were upregulated in R2 cells in the stationary phase. Wild-type and R2 cells showed similar heat shock responses at hsp mRNA and HSP levels; however, the σ32 level in the mutant was not downregulated in the shut-off stage. Strain R2 harbouring plasmid-borne dnaK grew at 37°C, did not accumulate HSPs, and was more sensitive to toluene than strain R2. It is worth to note that that revertant of R2 able to grow at 37°C were isolated and exhibited a replacement of Pro445 by Ser or Leu in DnaK. Thus, the mutation in dnaK causes the temperature-sensitive phenotype, improper stabilization of σ32 leading to HSP accumulation and increased toluene resistance in strain R2. [ABSTRACT FROM AUTHOR]

The article discusses a study which examines the crucial role of AP-1-like transcription factors in Neurospora crassa's oxidative stress responses and the function of NAP-1 in the regulation of the glutathione s-transferase expression. Findings reveal that AP-1-like transcription factor gene's elimination in Neurospora crassa could slightly rose its sensitivity to oxidative stressors. It indicates that the oxidative upregulation of several menadione-inducible genes (migs) were NAP-1 dependent.

The article discusses a study which examines the role of OS-2 mitogen activated protein kinase in regulating the clock-controlled gene ccg-1 in Neurospora crassa. Clock-controlled genes ccg-1, bli-3 and con-10 were induced by osmotic stress in an OS-2 dependent manner. Osmotic stress did not affect the expression of clock genes frq, wc-1 and wc-2 or of clock-controlled genes ccg-2 and bli-4. The findings suggest that OS-2 has a role in the regulation of certain circadian-clock output genes.

L-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, D-hydroxyproline dehydrogenase and Δ1-pyrroline-4-hydroxy- 2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. D-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (D-hydroxyproline) and some artificial electron acceptor( s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α4β4γ4), and two FADs, FMN, and [2Fe-2S] ironsulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the L-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on L-hydroxyproline (as well as D-hydroxyproline) but not L- and D-proline, indicating that this pathway is related only to L-hydroxyproline degradation, which is not linked to proline metabolism. [ABSTRACT FROM AUTHOR]

L-hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, D-hydroxyproline dehydrogenase and Δ(1)-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. D-hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (D-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α(4)β(4)γ(4)), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the L-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on L-hydroxyproline (as well as D-hydroxyproline) but not L- and D-proline, indicating that this pathway is related only to L-hydroxyproline degradation, which is not linked to proline metabolism.