Biomedical subjects

Elisabetta Zennaro

Publications and source records attributed to Elisabetta Zennaro.

9 recordsLinked to original sources

PubMed2007

Resistance to glycopeptide antibiotics in the teicoplanin producer is mediated by van gene homologue expression directing the synthesis of a modified cell wall peptidoglycan.

Glycopeptide resistance has been studied in detail in enterococci and staphylococci. In these microorganisms, high-level resistance is achieved by replacing the C-terminal D-alanyl-D-alanine of the nascent peptidoglycan with D-alanyl-D-lactate or D-alanyl-D-serine, thus reducing the affinities of glycopeptides for cell wall targets. Reorganization of the cell wall is directed by the expression of the van gene clusters. The identification of van gene homologs in the genomes of several glycopeptide-producing actinomycetes suggests the involvement of a similar self-resistance mechanism to avoid suicide. This report describes a comprehensive study of self-resistance in Actinoplanes teichomyceticus ATCC 31121, the producer of the clinically relevant glycopeptide teicoplanin. A. teichomyceticus ATCC 31121 showed a MIC of teicoplanin of 25 microg/ml and a MIC of vancomycin of 90 microg/ml during vegetative growth. The vanH, vanA, and vanX genes of A. teichomyceticus were found to be organized in an operon whose transcription was constitutive. Analysis of the UDP-linked peptidoglycan precursors revealed the presence of UDP-glycomuramyl pentadepsipeptide terminating in D-alanyl-D-lactate. No trace of precursors ending in d-alanyl-d-alanine was detected. Thus, the van gene complex was transcribed and expressed in the genetic background of A. teichomyceticus and conferred resistance to vancomycin and teicoplanin through the modification of cell wall biosynthesis. During teicoplanin production (maximum productivity, 70 to 80 microg/ml), the MIC of teicoplanin remained in the range of 25 to 35 microg/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 microg/ml.

Actinobacteria↗

PubMed2006

The Pseudomonas quorum-sensing regulator RsaL belongs to the tetrahelical superclass of H-T-H proteins.

In the opportunistic human pathogen Pseudomonas aeruginosa, quorum sensing (QS) is crucial for virulence. The RsaL protein directly represses the transcription of lasI, the synthase gene of the main QS signal molecule. On the basis of sequence homology, RsaL cannot be predicted to belong to any class of characterized DNA-binding proteins. In this study, an in silico model of the RsaL structure was inferred showing that RsaL belongs to the tetrahelical superclass of helix-turn-helix proteins. The overall structure of RsaL is very similar to the N-terminal domain of the lambda cI repressor and to the POU-specific domain of the mammalian transcription factor Oct-1 (Oct-1 POUs). Moreover, residues of Oct-1 POUs important for structural stability and/or DNA binding are conserved in the same positions in RsaL and in its homologs found in GenBank. These residues were independently replaced with Ala, and the activities of the mutated variants of RsaL were compared to that of the wild-type counterpart in vivo by complementation assays and in vitro by electrophoretic mobility shift assays. The results validated the RsaL in silico model and showed that residues Arg 20, Gln 38, Ser 42, Arg 43, and Glu 45 are important for RsaL function. Our data indicate that RsaL could be the founding member of a new protein family within the tetrahelical superclass of helix-turn-helix proteins. Finally, the minimum DNA sequence required for RsaL binding on the lasI promoter was determined, and our data support the hypothesis that RsaL binds DNA as a dimer.

Amino Acid Sequence↗

PubMed2006

The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter.

A mutation in the rsaL gene of Pseudomonas aeruginosa produces dramatically higher amounts of N-acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the rsaL-lasI bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the lasI promoter.

Bacterial Proteins↗

PubMed2005

An active-like structure in the unphosphorylated StyR response regulator suggests a phosphorylation- dependent allosteric activation mechanism.

StyR belongs to the FixJ subfamily of signal transduction response regulators; it controls transcription of the styABCD operon coding for styrene catabolism in Pseudomonas fluorescens ST. The crystal structure of unphosphorylated StyR is reported at 2.2 A resolution. StyR is composed of an N-terminal regulatory domain (StyR-N) and a C-terminal DNA binding domain (StyR-C). The two domains are separated by an elongated linker alpha helix (34 residues), a new feature in known response regulator structures. StyR-C is structured similarly to the DNA binding domain of the response regulator NarL. StyR-N shows structural reorganization of the phosphate receiving region involved in activation/homodimerization: specific residues adopt an "active-like" conformation, and the alpha4 helix, involved in dimerization of the homologous FixJ response regulator, is trimmed to just one helical turn. Overall, structural considerations suggest that phosphorylation may act as an allosteric switch, shifting a preexisting StyR equilibrium toward the active, dimeric, DNA binding form.

Allosteric Regulation↗

PubMed2005

Dual role of response regulator StyR in styrene catabolism regulation.

In Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and repressed by the addition of preferred carbon sources. PstyA is regulated by the StyS/StyR two-component system. The integration host factor (IHF) also plays a positive role in PstyA regulation. Three distinct StyR binding sites, which have different affinities for this response regulator, have been characterized on PstyA. The high-affinity StyR binding site (STY2) is necessary for promoter activity. The DNA region upstream of STY2 contains a lower-affinity StyR binding site, STY1, that partially overlaps the IHF binding site. Deletion of this region, designated URE (upstream regulatory element), has a dual effect on the PstyA promoter, decreasing the styrene-dependent activity and partially relieving the glucose repression. The lowest-affinity StyR binding site (STY3) is located downstream of the transcription start point. Deletion of the URE region and inactivation of the STY3 site completely abolished glucose-mediated repression of PstyA. In the proposed model StyR can act either as an activator or as a repressor, depending on which sites it occupies in the different growth conditions. We suggest that the cellular levels of phosphorylated StyR, as determined by StyS sensor kinase activity, and the interplay of this molecule with IHF modulate the activity of the promoter in different growth conditions.

Bacterial Proteins↗

PubMed2004

CO sniffing through heme-based sensor proteins.

O(2), NO, and CO are substrates, products, and/or modulators in many enzymatic reactions including O(2) respiration, denitrification, N(2) fixation, and methanogenesis. Over the last decade, O(2), NO, and CO have been reported to act as signaling molecules in the control of several distinct biological functions. Here, the heme-based mechanisms of CO sensing by the bacterial homodimeric CooA and the mammalian heterodimeric NPAS2-BMAL1 transcriptional factors are reviewed, and the signal transduction pathways are discussed.

Animals↗

PubMed2003

Styrene-catabolism regulation in Pseudomonas fluorescens ST: phosphorylation of StyR induces dimerization and cooperative DNA-binding.

Styrene is an important chemical extensively used in the petrochemical and polymer industries. In Pseudomonas fluorescens ST, styrene metabolism is controlled by a two-component regulatory system, very uncommon in the degradation of aromatic compounds. The two-component regulatory proteins StyS and StyR regulate the expression of the styABCD operon, which codes for styrene degradation. StyS corresponds to the sensor kinase and StyR to the response regulator, which is essential for the activation of PstyA, the promoter of the catabolic operon. In two-component systems, the response regulator is phosphorylated by the cognate sensor kinase. Phosphorylation activates the response regulator, inducing DNA-binding. The mechanism underlying this activation has been reported only for a very few response regulators. Here, the effect of phosphorylation on the oligomeric state and on the DNA-binding properties of StyR has been investigated. Phosphorylation induces dimerization of StyR, the affinity of dimeric StyR for the target DNA is higher than that of the monomer, moreover dimeric StyR binding to the DNA target is cooperative. Furthermore, StyR oligomerization may be driven by the DNA target. This is the first direct demonstration that StyR response regulator binds to the PstyA promoter.

Bacterial Proteins↗

PubMed2002

Occurrence and properties of glutathione S-transferases in phenol-degrading Pseudomonas strains.

Pseudomonas sp. strains, able to degrade aromatic compounds such as phenol, were chosen to investigate the occurrence and characteristics of glutathione S-transferases (GSTs). Affinity chromatography purification showed the presence of at least one GST in each studied strain. The purified proteins exhibited a great variety in the N-terminal sequences and different enzyme activities with the standard GST substrates tested. Two Pseudomonas strains, M1 and CF600, were chosen to investigate the GST activities under different growth conditions. Therefore, cells were grown either on phenol or on different nonaromatic carbon sources in the presence and absence of increasing phenol concentrations. In strain M1 a strong correlation between the activities of the catechol 1,2-dioxygenase and GST was observed in all the tested conditions. Moreover, growth on different organic acids also affected GST activity levels, with a negative correlation with the specific growth rate determined by each substrate. These results suggest a possible function of GST as a response to specific metabolic conditions determined by phenol toxicity and/or catabolism and the metabolic status of the cells. The same experiments performed with the CF600 strain did not show induction of GST activity in any of the tested conditions, indicating that GST_CF600 probably has a different role in cell metabolism. Native gel electrophoresis gave indications that GST dimerization could be an important process in the modulation of GST activity.

Amino Acid Sequence↗

PubMed2002

Integration host factor is essential for the optimal expression of the styABCD operon in Pseudomonas fluorescens ST.

The StyS/StyR two-component regulatory system of Pseudomonas fluorescens ST controls the expression of the styABCD operon coding for the styrene degradation upper pathway. In a previous work we showed that the promoter of the catabolic operon (PstyA) is induced by styrene and repressed to differing extents by organic acids or carbohydrates. In order to study the mechanisms controlling the expression of this operon, we performed a functional analysis on 5' deletions of PstyA by the use of a promoter-probe system. These studies demonstrated that a palindromic region (sty box), located from nucleotides -52 to -37 with respect to the transcriptional start point is essential for PstyA activity. Moreover, additional regulatory regions involved in the modulation of PstyA activity were found along the promoter sequence. In particular, deletion of a putative StyR binding site, homologous to the 3' half of the sty box and located upstream of this box, resulted in 65% reduction of the induction level of the reporter gene. Additionally, we performed bandshift assays with a DNA probe corresponding to PstyA and protein crude extracts from P. fluorescens ST, using specific DNA fragments as competitors. In these experiments we demonstrated that IHF binds an AT-rich region located upstream of the sty box. On the basis of this finding, coupled with the results obtained with PstyA functional analysis, we suggest that the role of the IHF-mediated DNA bend is to bring closer, in an overlapping position, the upstream StyR putative binding site and the downstream sty box, and that the formed complex enhances transcription.

Bacterial Proteins↗