The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 ??-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.     

Significant contribution of the pgdA gene to the virulence of Streptococcus suis

Streptococcus suis is a major swine pathogen and emerging zoonotic agent. In this study we have determined the muropeptide composition of S. suis peptidoglycan (PG) and found, among other modifications, N-deacetylated compounds. Comparison with an isogenic mutant showed that the product of the pgdA gene is responsible for this specific modification which occurred in very low amounts. Low level of PG N-deacetylation correlated with absence of significant lysozyme resistance when wild-type S. suis was grown in vitro. On the other hand, expression of the pgdA gene was increased upon interaction of the bacterium with neutrophils in vitro as well as in vivo in experimentally inoculated mice, suggesting that S. suis may enhance PG N-deacetylation under these conditions. Evaluation of the DeltapgdA mutant in both the CD1 murine and the porcine models of infection revealed a significant contribution of the pgdA gene to the virulence traits of S. suis. Reflecting a severe impairment in its ability to persist in blood and decreased ability to escape immune clearance mechanisms mediated by neutrophils, the DeltapgdA mutant was highly attenuated in both models. The results of this study suggest that modification of PG by N-deacetylation is an important factor in S. suis virulence.     

Use of Selective Capture of Transcribed Sequences To Identify Genes Preferentially Expressed by Streptococcus suis upon Interaction with Porcine Brain Microvascular Endothelial Cells

By using the selective capture of transcribed sequences (SCOTS) approach, we identified 28 genes preferentially expressed by the major swine pathogen and zoonotic agent Streptococcus suis upon interaction with porcine brain microvascular endothelial cells. Several of these genes may be considered new S. suis candidate virulence factors. Results from this study demonstrate the suitability of SCOTS for the elucidation of gene expression in streptococcal species and may contribute to a better understanding of the pathogenesis of S. suis infections.     

Single-crystal EPR study at 95 GHz of the type 2 copper site of the inhibitor-bound quercetin 2,3-dioxygenase

An electron-spin-echo-detected, electron-paramagnetic-resonance study has been performed on the type 2 copper site of quercetin 2,3-dioxygenase from Aspergillus japonicus. In the protein, copper is coordinated by three histidine nitrogens and two sulfurs from the inhibitor diethyldithiocarbamate. A single crystal of the protein was studied at 95 GHz and the complete g-tensor determined. The electron-paramagnetic-resonance data are compatible with two orientations of the principal g-axes in the copper center, one of which is preferred on the basis of an analysis of the copper coordination and the d-orbitals that are involved in the unpaired-electron orbital. For this orientation, the principal z-axis of the g-tensor makes an angle of 19 degrees with the Cu-N(His112) bond and the N of His112 may be considered the axial ligand. The singly occupied molecular orbital contains a linear combination of copper dxy and dyz-orbitals, which are antibonding with atomic orbitals of histidine nitrogens and diethyldithiocarbamate sulfurs. The orientation of the g-tensor for the quercetin 2,3-dioxygenase is compared with that for type 1 copper sites.     

Reconstitution of the type-1 active site of the H145G/A variants of nitrite reductase by ligand insertion

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.     

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms

Aims: It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. Methods and Results: It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3′‐[1‐[(phenylamino)carbonyl]‐3,4‐tetrazolium]Bis(4‐methoxy)‐6‐nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. Conclusions: We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. Significance and Impact of the Study: This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.