Physiological modulation of the sigma54-dependent promoter Pu of the TOL plasmid: negative regulatory role of the TurA protein of Pseudomonas putida in the response to suboptimal growth temperatures

The Pseudomonas putida sigma54-dependent Pu promoter of the TOL upper operon, involved in toluene utilization, represents an optimal reference model to investigate the complexity of the regulatory networks for aromatic substances. Indeed, Pu activation in the presence of aromatic effectors is finely tuned in response to several factors connected to cell physiology and environmental physiochemical conditions. The current model for Pu activation includes several details about the molecular switch involved in toluene response. However, the molecular mechanisms of the fine physiological regulation remain to be fully understood. We identified, using a reverse-genetic approach, a small Pu-binding protein, TurA, and we present evidence that TurA-Pu DNA interactions could participate in direct negative modulation of Pu activity both in vivo and in vitro. Furthermore, we present evidence that TurA-mediated repression of Pu activity is further increased at suboptimal growth temperatures and that induction of TOL upper operon at suboptimal temperatures is growth limiting. Our results show that TurA cellular concentrations are growth-phase and temperature regulated. We suggest that the role of TurA could be limitation of the expression of the toluene-degrading enzymes under suboptimal growth conditions to prevent toxic effects. Our in silico analysis indicated that TurA and, possibly, the other members of the MvaT family are structurally related to the nucleoid-associated protein H-NS of Escherichia coli. It can be suggested that the members of the MvaT family have roles in Pseudomonas species analogous to H-NS-like proteins in other Gram-negative species as global modulators of gene expression

Identification of genes regulated by the MvaT-like paralogues TurA and TurB of Pseudomonas putida KT2440

The genome of the Pseudomonas putida strain KT2440 contains five paralogous proteins (TurA, TurB, TurC, TurD and TurE) of the H-NS-like MvaT class of transcription regulators. TurA and TurB belong to groups I and II, respectively, both containing orthologous MvaT proteins that are present in all Pseudomonadaceae species. On the contrary, TurC, TurD and TurE belong to group III, which contains species-specific paralogous MvaT proteins. We analysed the global effects on the P. putida KT2440 transcriptome of eliminating the conserved TurA and TurB proteins, which had been identified in our previous studies aimed to search for novel specific co-regulators of the upper TOL operon for toluene biodegradation. While the loss of TurA de-repressed the expression of many genes covering a broad range of functional classes in both mid-exponential and early stationary phases, the absence of TurB brought about a very different outcome. Although the loss of TurB affected also very different functions, the number of genes that changed in the turB mutant was fivefold smaller than that of TurA. Furthermore, TurB does not act generally as repressor. Interestingly, the degree of overlap between their mutual regulons is very limited. A closer examination of one case where such overlap clearly occurs (a gene cluster for biosynthesis of lipodepsinonapeptide phytotoxins) revealed that TurA and TurB can act in concert, perhaps by forming a heterodimer. In addition, our results indicate that TurA is the master regulator of TurB as well as of the other paralogues, TurD and TurE

Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and -xylene isolated from a polycyclic aromatic hydrocarbon-contaminated soil

Rhodococcus opacus R7 was isolated from a soil contaminated with polycyclic aromatic hydrocarbons for its ability to grow on naphthalene. The strain was also able to degrade o-xylene, the isomer of xylenes most recalcitrant to microbial degradation. The catabolic pathways for naphthalene and o-xylene were investigated by identification of metabolites in R. opacus R7 cultures performed with the two hydrocarbons and by evaluation of some enzymes involved in the metabolism of these compounds. 1,2-Dihydro-1,2-dihydroxynaphthalene, salicylic and gentisic acids were identified as metabolites in cultures exposed to naphthalene. This suggests that the degradation occurs through the dioxygenation of the aromatic ring with the formation of 1,2-dihydro-1,2-dihydroxynaphthalene, dehydrogenated to the corresponding 1,2-dihydroxy derivative which is further oxidized to salicylic acid, a key intermediate of naphthalene metabolism; this compound is converted to gentisic acid cleaved by a gentisate 1,2-dioxygenase. From R. opacus R7 cultures supplied with o-xylene, 2,3-dimethylphenol and 3,4-dimethylcatechol were observed. The pathway of o-xylene involves the monooxygenation of the benzene nucleus leading to dimethylphenol which is further metabolised to 3,4-dimethylcatechol, followed by a meta cleavage reaction, catalyzed by the catechol 2,3-dioxygenase. R. opacus R7 is the first strain thus far described both in Gram-negative and Gram-positive bacteria which has the ability to degrade both a polycyclic aromatic hydrocarbon such as naphthalene and a monocyclic aromatic hydrocarbon such as o-xylene

Novel physiological modulation of the Pu promoter of TOL plasmid : negative regulatory role of the TurA protein of Pseudomonas putida in the response to sub-optimal growth temperatures

From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TurA, able to bind to DNA fragments bearing the entire Pu promoter sequence of the TOL plasmid. The knock-out inactivation of the turA gene resulted in enhanced transcription initiation from the Pu promoter, initially suggesting a negative regulatory role of TurA on Pu expression. Ectopic expression of TurA both in P. putida and in Escherichia coli reporter strains and transcription in vitro of the Pu promoter in the presence of purified TurA confirmed the TurA repressor role on Pu activity. turA gene inactivation did not significantly alter two well characterized physiological regulations of the Pu expression in routine conditions of cultivation, exponential silencing, and carbon-mediated repression, respectively. However, the growth at suboptimal temperatures resulted in a TurA-dependent increase of Pu repression. These results strongly suggest that a physiological significance of the negative role of TurA on Pu activity could be limitation of the expression of the toluene-degrading enzymes at suboptimal growth temperatures. Therefore, the identification of TurA as Pu-binding protein revealed a novel physiological modulation of Pu promoter that is different from those strictly nutritional described previously

La biopsia minima dei noduli mammari in fase preclinica

In conjuction with ultrasonography, mammography is the most common instrumental method used in the screening program of subclinical breast lesions. The Authors present their method of preoperative localization and minimal biopsy of subclinical breast lesions. According to our experience, the most efficient technique is founded on marking the suspected area, guided by mammography, with vegetal coal, that remain 'in situ' about 2 weeks. We have performed this procedure in 40 cases. A diagnosis positive for carcinoma was made in 9 cases. Hospitalization of the patients is not necessary: in fact marked lesion is excised under local anaesthesia and the whole operation takes about 30 minutes. Complications have been negligible. The aesthetic result is agreeable and the patients acceptance of a minimal biopsy is good

Autoimmunity and thyroid function in patients with chronic active hepatitis treated with recombinant interferon alpha-2a

The occurrence of thyroid abnormalities and the appearance of organ- and non-organ-specific autoantibodies during long-term recombinant interferon alpha-2a (IFN-alpha) therapy were studies in 86 and 51 consecutive outpatients with hepatitis C and B virus-related chronic active hepatitis (CAH-HCV and CAH-HBV), respectively. Most patients had longstanding community-acquired hepatitis. At baseline, 9.3% of CAH-HCV and 3.9% of CAH-HBV patients showed clinical and/or biochemical signs of thyroid dysfunction. The remaining patients were euthyroid, although anti-thyroid autoantibodies were found in 33/78 (42.3%) of CAH-HCV and in 5/49 (10.2%) of CAH-HBV patients. During IFN-alpha treatment, increased anti-thyroid autoantibody levels were seen in 40% of CAH-HCV initially negative patients, while they became detectable in no more than 10% of CAH-HBV patients. Interferon-alpha-induced hypo- or hyperthyroidism was recorded in 12 of 35 CAH-HCV patients treated for 12 months (34.3%). Only one CAH-HBV patient developed hyperthyroidism. High titers of anti-nuclear autoantibodies (ANA) were recorded at enrolment in 5/36 (13.8%) of CAH-HCV and in 3/16 (18.7%) of CAH-HBV patients. Only one CAH-HCV patient displayed anti-parietal cell antibodies (PCA). After IFN-alpha treatment, ANA were found in 10/28 (35.7%) and PCA in 2/28 (7.1%) of CAH-HCV patients, while an additional CAH-HBV patient developed PCA. but not ANA. However, no signs of systemic autoimmune disease were recorded. In conclusion. more than half of the patients with chronic active hepatitis C, but only one-tenth of those with hepatitis B, displayed thyroid- and/or non-organ-specific autoantibodies prior to or during treatment with IFN-alpha. As most of the antibody-positive patients developed permanent thyroid disorders during IFN-alpha therapy, the risk of development of organ-specific autoimmunity should be assessed carefully and incorporated in the cost/effectiveness analysis in patients with longstanding hepatitis who are candidates for IFN-alpha treatment