Role of PumB antitoxin as a transcriptional regulator of the PumAB type-II toxin-antitoxin system and its endoribonuclease activity on the PumA (toxin) transcript.

The PumAB type-II toxin-antitoxin (TA) system is encoded by pumAB genes that are organized into an operon. This system is encoded by the pUM505 plasmid, isolated from a Pseudomonas aeruginosa clinical strain. The pumA gene encodes a putative RelE toxin protein (toxic component), whereas the pumB gene encodes a putative HTH antitoxin protein. The expression of the PumAB system in Escherichia coli confers plasmid stability. In addition, PumA toxin overexpression in P. aeruginosa possesses the capability to increase bacterial virulence, an effect that is neutralized by the PumB antitoxin. The aim of this study was to establish the mechanism of regulation of the PumAB toxin-antitoxin system from pUM505. By an in silico analysis of the putative regulatory elements, we identified two putative internal promoters, PpumB and PpumB-AlgU (in addition to the already reported PpumAB), located upstream of pumB. By RT-qPCR assays, we determined that the pumAB genes are transcribed differentially, in that the mRNA of pumB is more abundant than the pumA transcript. We also observed that pumB could be expressed individually and that its mRNA levels decreased under oxidative stress, during individual expression as well as co-expression of pumAB. However, under stressful conditions, the pumA mRNA levels were not affected. This suggests the negative regulation of pumB by stressful conditions. The PumB purified protein was found to bind to a DNA region located between the PpumAB and the pumA coding region, and PumA participates in PumB binding, suggesting that a PumA-PumB complex co-regulates the transcription of the pumAB operon. Interestingly, the pumA mRNA levels decreased after incubation in vitro with PumB protein. This effect was repressed by ribonuclease inhibitors, suggesting that PumB could function as an RNAse toward the mRNA of the toxin. Taken together, we conclude that the PumAB TA system possesses multiple mechanisms to regulate its expression, as well as that the PumB antitoxin generates a decrease in the mRNA toxin levels, suggesting an RNase function. Our analysis provides new insights into the understanding of the control of TA systems from mobile plasmid-encoded genes from a human pathogen.

Plasmid pUM505 encodes a Toxin-Antitoxin system conferring plasmid stability and increased Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin-Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET-pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans, indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.

Genes from pUM505 plasmid contribute to Pseudomonas aeruginosa virulence.

The pUM505 plasmid was isolated from a clinical strain of Pseudomonas aeruginosa. This plasmid contains a genomic island with sequence similar to islands found in chromosomes of virulent P. aeruginosa clinical isolates. The objective of this work was to determine whether pUM505 increases the virulence of P. aeruginosa and to identify the genes responsible for this property. First, using the lettuce-leaf model, we found that pUM505 significantly increases the virulence of P. aeruginosa reference strain PAO1. pUM505 also increased the PAO1 virulence in a murine model and increased cytotoxicity of this strain toward HeLa cells. Thus, we generated a pUM505 gene library of 103 clones in the pUCP20 binary vector. The library was transferred to Escherichia coli TOP10 and P. aeruginosa PAO1 to identify genes. The lettuce-leaf model allowed us to identify three recombinant plasmids that increased the virulence of both E. coli and P. aeruginosa strains. These recombinant plasmids also increased the virulence of the PAO1 strain in mice and induced a cytotoxic effect in HeLa cells. Eleven genes were identified in the virulent transformants. Of these genes, only the pUM505 ORF 2 has homology with a gene previously implicated in virulence. These results indicate that pUM505 contains several genes that encode virulence factors, suggesting that the plasmid may contribute directly to bacterial virulence.

Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes.

We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.

Molecular analysis of an NAD-dependent alcohol dehydrogenase from the zygomycete Mucor circinelloides.

NAD-dependent alcohol dehydrogenase (ADH) activity was detected mainly in the cytosol of aerobically cultured mycelium and in anaerobically grown yeast cells of Mucor circinelloides. ADH levels were about 2.5-fold higher in yeast cells than in mycelium; zymogram analysis suggested that the same ADH enzyme is produced in both developmental stages. The enzyme, named ADH1, was purified to homogeneity from yeast cells, using ion-exchange and affinity chromatography. The active ADH1 appears to be a homomeric tetramer of 37,500-kDa subunits. Km values obtained for acetaldehyde, ethanol, NADH and NAD+ indicated that in vivo the enzyme mainly serves to reduce acetaldehyde to ethanol. Amino acid sequences of internal peptides obtained from the purified ADH1 were used to design oligonucleotides that allowed the cloning of the corresponding cDNA by RT-PCR, and the characterization of the genomic DNA sequence. The adh1 ORF is interrupted by two small introns located towards the 5'-end. M. circinelloides adh1 encodes a protein of 348 amino acids, which display moderate to high overall identity to several hypothetical ADH enzymes from the related zygomycete Rhizopus oryzae. adh1 mRNA is expressed at similar levels in aerobic mycelium and anaerobic yeast cells. During exponential growth under aerobic conditions, the level of adh1 transcript was correlated with the glucose concentration in the growth medium.