Molecular Characterization and Identification of Bacillus clausii Strains Marketed for Use in Oral Bacteriotherapy

A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillus spores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than with Bacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains of B. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.     

Role of the extracellular loop in the folding of a CFTR transmembrane helical hairpin

The folding of membrane-spanning domains into their native functional forms depends on interactions between transmembrane (TM) helices joined by covalent loops. However, the importance of these covalent linker regions in mediating the strength of helix-helix associations has not been systematically addressed. Here we examine the potential structural impact of cystic fibrosis-phenotypic mutations in the extracellular loop 2 (ECL2) on interactions between the TM3 and TM4 helices of the cystic fibrosis transmembrane conductance regulator (CFTR) in constructs containing CFTR residues 194-241. When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R) were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild-type position and that decreased migration rates correlate with increasing hairpin alpha-helical content as measured by circular dichroism spectra in sodium dodecyl sulfate micelles. The decreased mobility of TM3/4 constructs by introduction of non-native residues is interpreted in terms of an elongation or "opening" of the helical hairpin and concomitant destabilization of membrane-based helix-helix interactions. Our results support a role for short loop regions in dictating the stability of membrane protein folds and highlight the interplay between membrane-embedded helix-helix interactions and loop conformation in influencing the structure of membrane proteins.     

Linear and cyclic peptides as substrates for Lyn tyrosine kinase

Two Tyr residues are supposed to play a crucial role in the regulation of protein tyrosine kinases of the Src family. Autophosphorylation of Src Tyr416 correlates with enzyme activation, while phosphorylation of C-terminal Tyr527 by Csk gives rise to inactive forms of Src kinases. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogenelyndisplays a particularly high affinity (K_m20 μm ) toward the dimeric linear and cyclic derivatives of the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH which reproduces the main autophosphorylation site of most of the Src enzymes. Under the experimental conditions used only one Tyr residue of the dimeric sequence can be phosphorylated [P. Ruzza, A. Calderan, B.Filippi, B. Biondi, A. Donella Deana, L. Cesaro, L. A. Pinna & G. Borin (1995) Int. J. Peptide Protein Res. 45, 529–539]. The present study addresses the problem of the efficiency displayed by Lyn towards the two Tyr residues located at positions 5 and 12 of the dimeric peptide. To this purpose, two tetradecapeptides were synthesized by the classical solution method, each containing one of the two Tyr residues alternatively replaced by Phe, and the corresponding univocal cyclic form. A possible correlation between the different structural properties induced by the modifications of the native sequence and the ability of the peptides to act as Lyn substrates was noted. The kinetic data obtained indicate that Lyn phosphorylates the residues located at different positions in the two linear analogues differently. In particular, while the Tyr5, Phe12 derivative presents aK_mvalue similar to those obtained for the dimeric linear and cyclic unmodified analogues, theK_mvalue of the Phe5, Tyr12 derivative is two-fold higher than those found for the above-mentioned peptides. Moreover, as previously reported for the linear and cyclic dimeric forms of the native sequence, in the mono-tyrosine containing series of dimers the still conformationally flexible cyclic derivative shows a phosphorylation efficiency two-fold higher than those found for the linear derivatives. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

The response of Escherichia coli biofilm to salicylic acid

In this research, salicylic acid is proposed as an alternative biocide-free agent suitable for a preventive or integrative anti-biofilm approach. Salicylic acid has been proved to: (1) reduce bacterial adhesion up to 68.1 ± 5.6%; (2) affect biofilm structural development, reducing viable biomass by 97.0 ± 0.7% and extracellular proteins and polysaccharides by 83.9 ± 2.5% and 49.5 ± 5.5% respectively; and (3) promote biofilm detachment 3.4 ± 0.6-fold. Moreover, salicylic acid treated biofilm showed an increased amount of intracellular (2.3 ± 0.2-fold) and extracellular (2.1 ± 0.3-fold) reactive oxygen species, and resulted in increased production of the quorum sensing signal indole (7.6 ± 1.4-fold). For the first time, experiments revealed that salicylic acid interacts with proteins that play a role in quorum sensing, reactive oxygen species accumulation, motility, extracellular polymeric matrix components, transport and metabolism.     

Crystal structure of the cytomegalovirus DNA polymerase subunit UL44 in complex with the C terminus from the catalytic subunit. Differences in structure and function relative to unliganded UL44

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the "connector loop" of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic "plug" from the UL54 peptide and Ile(135) of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-to-head dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44.     

Towards the identification of the allosteric Phe-binding site in phenylalanine hydroxylase

The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe–PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant.