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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]

OxyR regulates the expression of a peroxiredoxin (AhpC) and two catalases (KatA and KatB), which play roles in peroxide protection, at the transcription level in Pseudomonas putida KT2442. Proteome analysis indicated significantly increased amounts of the enzymes AhpC, KatA, KatB, and a peroxiredoxin reductase (AhpF) in the oxyR1 mutant cells; these increases reflected the upregulation of the expression of the genes encoding these enzymes. Additionally, although the effect of oxyR1 mutation on the trxB transcript level was not clearly evident, it increased the amount of thioredoxin reductase (TrxB) by fivefold. Primer extension analysis revealed that trxB was constitutively transcribed from the P1 site; however, hydrogen peroxide treatment lowered the transcription of trxB from P1 but induced its transcription from P2. Adjacent to the −35 base of the P2 initiation site, sequences similar to those involved in the proposed OxyR binding in Escherichia coli were found in a region to which OxyR was shown to bind. These observations suggest that in P. putida, OxyR regulates TrxB expression by promoting trxB transcription from the P2 site when oxidative stresses lowered the transcription from the constitutive P1 site. [ABSTRACT FROM AUTHOR]

OxyR is known to activate/repress the expression of the oxyR regulon, which consists of several genes, which play important antioxidant role in Escherichia coli. To elucidate the role of OxyR in Pseudomonas putida KT2442, the oxyR1 mutation that caused the upregulation of ahpC in a toluene-resistant variant strain was introduced, because no null mutants in oxyR were isolated. This mutation was shown to cause the accumulation of a catalase (KatA) along with AhpC throughout the growth, and of a RpoS-dependent catalase/peroxidase (KatB) in the stationary phase. Following the identification of the transcription start site of two catalase genes, sequences similar to those involved in the proposed OxyR binding for E. coli were found upstream from each of the promoter regions of katA and katB, as well as ahpC. Purified OxyR was shown to bind to these sequences, under both reduced and oxidized states. Moreover, the oxyR1 mutation increased the transcription levels of these genes. These results are consistent with the conclusion, distinct from those observed in an opportunistic pathogen Pseudomonas aeruginosa, that OxyR controlled expression of all the principal peroxide-degrading enzymes in P. putida. The mutation did not cause any notable changes in the transcriptional levels of several antioxidant genes, including those of glutathione reductase, glutaredoxins and thioredoxins, which would involve maintenance of the cellular thiol-disulfide balance, suggesting that the transcriptional regulation of these antioxidant genes should be different from that of katA, katB and ahpC in P. putida. [ABSTRACT FROM AUTHOR]