Michele Masulli

Glutathione transferases in bacteria

Bacterial glutathione transferases (GSTs) are part of a superfamily of enzymes that play a key role in cellular detoxification. GSTs are widely distributed in prokaryotes and are grouped into several classes. Bacterial GSTs are implicated in a variety of distinct processes such as the biodegradation of xenobiotics, protection against chemical and oxidative stresses and antimicrobial drug resistance. In addition to their role in detoxification, bacterial GSTs are also involved in a variety of distinct metabolic processes such as the biotransformation of dichloromethane, the degradation of lignin and atrazine, and the reductive dechlorination of pentachlorophenol. This review article summarizes the current status of knowledge regarding the functional and structural properties of bacterial GSTs.

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications

BACKGROUND Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Escherichia coli in Europe: an overview.

Escherichia coli remains one of the most frequent causes of several common bacterial infections in humans and animals. E. coli is the prominent cause of enteritis, urinary tract infection, septicaemia and other clinical infections, such as neonatal meningitis. E. coli is also prominently associated with diarrhoea in pet and farm animals. The therapeutic treatment of E. coli infections is threatened by the emergence of antimicrobial resistance. The prevalence of multidrug-resistant E. coli strains is increasing worldwide principally due to the spread of mobile genetic elements, such as plasmids. The rise of multidrug-resistant strains of E. coli also occurs in Europe. Therefore, the spread of resistance in E. coli is an increasing public health concern in European countries. This paper summarizes the current status of E. coli strains clinically relevant in European countries. Furthermore, therapeutic interventions and strategies to prevent and control infections are presented and discussed. The article also provides an overview of the current knowledge concerning promising alternative therapies against E. coli diseases.

Die for the community: an overview of programmed cell death in bacteria

Programmed cell death is a process known to have a crucial role in many aspects of eukaryotes physiology and is clearly essential to their life. As a consequence, the underlying molecular mechanisms have been extensively studied in eukaryotes and we now know that different signalling pathways leading to functionally and morphologically different forms of death exist in these organisms. Similarly, mono-cellular organism can activate signalling pathways leading to death of a number of cells within a colony. The reason why a single-cell organism would activate a program leading to its death is apparently counterintuitive and probably for this reason cell death in prokaryotes has received a lot less attention in the past years. However, as summarized in this review there are many reasons leading to prokaryotic cell death, for the benefit of the colony. Indeed, single-celled organism can greatly benefit from multicellular organization. Within this forms of organization, regulation of death becomes an important issue, contributing to important processes such as: stress response, development, genetic transformation, and biofilm formation.

Proteus mirabilis glutathione S-transferase B1-1 is involved in protective mechanisms against oxidative and chemical stresses.

We investigated the effects of several xenobiotics, including antimicrobial agents and general stress factors such as starvation, heat and osmotic shock, on the modulation of expression of Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1). The level of expression of PmGST B1-1 was established by both Western- and Northern-blot experiments. Our results show that several compounds can modulate expression of PmGST B1-1. The level of PmGST B1-1 increased when bacterial cells were exposed to a variety of stresses such as 1-chloro-2,4-dinitrobenzene, H(2)O(2), fosfomycin or tetracycline. A knock-out gst B gene was also constructed using the suicide vector pKNOCKlox-Ap. Successful inactivation of the wild-type gene was confirmed by PCR, DNA sequence analysis and Western blotting. Under normal culture conditions, this mutant was viable and displayed no significant phenotypic differences compared with the wild-type. However, viability tests revealed that the null mutant was more sensitive to oxidative stress in the form of H(2)O(2) and to several antimicrobial drugs when compared with the wild-type. These results suggest that PmGST B1-1 has an active role in the protection against oxidative stress generated by H(2)O(2) and it appears to be involved in the detoxification of antimicrobial agents.

Site‐directed mutagenesis of the Proteus mirabilis glutathione transferase B1‐1 G‐site

In order to investigate the roles of near N‐terminus Tyr, Cys, and Ser residues in the activity of bacterial glutathione transferase (GSTB1‐1) site‐directed mutagenesis was used to replace the following residues: Tyr‐4, Tyr‐5, Ser‐9, Cys‐10, Ser‐11, and Ser‐13. The results presented here show that, unlike all other alpha, mu, pi, theta and sigma classes of glutathione transferases so far investigated, GSTB1‐1 does not utilise any Tyr, Ser or Cys residue to activate glutathione. These results also suggest that the bacterial glutathione transferases may require classification into their own class.

Functional analysis of the evolutionarily conserved proline 53 residue in Proteus mirabilis glutathione transferase B1‐1

The role of the evolutionarily conserved residue Pro‐53 in Proteus mirabilis glutathione transferase B1‐1 has been examined by replacing it with a serine residue using site‐directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro‐53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.

Distribution of glutathione transferases in Gram-positive bacteria and Archaea.

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.

Evaluation of the role of two conserved active-site residues in beta class glutathione S-transferases.

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His(106) and Lys(107) of PmGST B1-1 to investigate their possible role in the enzymes catalytic activity. His(106) was mutated to Ala, Asn and Phe, and Lys(107) to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His(106) and Lys(107) in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.

Role of Ser11 in the stabilization of the structure of Ochrobactrum anthropi glutathione transferase

GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the K(m) for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.