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PubMed2001

Structure, specificity, and mode of interaction for bacterial albumin-binding modules.

We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.

Amino Acid Sequence↗
PubMed1991

ymoA, a Yersinia enterocolitica chromosomal gene modulating the expression of virulence functions.

The virulence functions of Yersinia enterocolitica include the pYV-encoded Yop proteins and YadA adhesin as well as the chromosome-encoded enterotoxin, Yst. The yop and yadA genes form a temperature-activated regulon controlled by the transcriptional activator VirF. Gene virF, also localized on pYV, is itself thermoinduced in the absence of other pYV genes. The enterotoxin yst gene is silent in some collection strains including strain W22703. This paper describes two Tn5-Tc1 chromosomal insertion mutants of W22703 transcribing virF, and hence the yop and yadA genes, at low temperature. These mutants also resumed their production of Yst, with its typical temperature dependence. Both mutations were insertions in the same gene called ymoA for 'Yersinia modulator'. The cloned ymoA gene fully complemented the two mutations. Several properties of the mutants suggest that ymoA encodes a histone-like protein. According to the nucleic acid sequence, the product of ymoA is an 8064 Da protein rich in aspartic acid (9%), glutamic acid (9%) and lysine (10.5%), but the predicted amino acid sequence shows no similarity with any described histone-like protein. This work supports recent reports which propose a role for DNA topology and bacterial chromatin structure in thermoregulation of virulence functions.

Amino Acid Sequence↗
PubMed2005

Peptides selected for binding to a virulent strain of Haemophilus influenzae by phage display are bactericidal.

Nontypeable Haemophilus influenzae (NTHi) is an obligate parasite of the oropharynx of humans, in whom it commonly causes mucosal infections such as otitis media, sinusitis, and bronchitis. We used a subtractive phage display approach to affinity select for peptides binding to the cell surface of a novel invasive NTHi strain R2866 (also called Int1). Over half of the selected phage peptides tested were bactericidal toward R2866 in a dose-dependent manner. Five of the clones encoded the same peptide sequence (KQRTSIRATEGCLPS; clone hi3/17), while the remaining four clones encoded unique peptides. All of the bactericidal phage peptides but one were cationic and had similar physical-chemical properties. Clone hi3/17 possessed a similar level of activity toward a panel of clinical NTHi isolates and H. influenzae type b strains but lacked bactericidal activity toward gram-positive (Enterococcus faecalis, Staphylococcus aureus) and gram-negative (Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella enterica) bacteria. These data indicate that peptides binding to bacterial surface structures isolated by phage display may prove of value in developing new antibiotics.

Amino Acid Sequence↗
PubMed1985

The effect of phospholipids from Listeria monocytogenes and Aspergillus fumigatus on degradation of bacterial antigens by mouse peritoneal macrophages.

Investigations were carried out on an influence of phospholipids from L. monocytogenes and A. fumigatus on the degradation of Streptococcus faecalis cell walls by mouse peritoneal macrophages. It has been found, that macrophages from mice stimulated with phospholipids under studies degraded streptococcal cell walls labeled with DL-alanine-I-14C more efficiently than normal macrophages. Activated macrophages degraded also more rapidly antigenic structures of bacterial cell walls reacting with specific fluorescein-labeled antibodies. Moreover, the increase in activity of lysosomal acid hydrolases in stimulated macrophages was observed. The experiments performed suggest that increased ability of macrophages to degradation of bacterial antigens may be connected with a primary augmentation of lysosomal enzyme activity.

Animals↗
PubMed2000

Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

Acarbose↗
PubMed2002

Culture-independent microbial community analysis reveals that inulin in the diet primarily affects previously unknown bacteria in the mouse cecum.

Inulin is a well-known fructose-based prebiotic which has been shown to stimulate the growth of bifidobacteria, a bacterial group generally considered beneficial for intestinal health. In the present study, we analyzed inulin-associated shifts in the total bacterial community of wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene by using DNA-based approaches independent of bacterial culturability. Mice were fed a high-fat, nonfiber diet with or without inulin inclusion at a 10% (wt/wt) concentration. Cecal contents were analyzed after 0, 3, and 9 weeks on the experimental diets. Inulin inclusion significantly affected the total bacterial community structure of the cecum as determined by both a nonselective percent-guanine-plus-cytosine-based profiling analysis and a more specific 16S ribosomal DNA sequence analysis. The shifts included stimulation of bifidobacteria and suppression of clostridia, but sequence comparison revealed that the major shifts were within previously unknown bacterial taxa. Concomitantly, significantly higher bacterial densities, determined by flow cytometry, were observed with the inulin-amended diet, and the metabolism of the cecal bacterial community was altered, as indicated by higher levels of residual short-chain fatty acids, particularly lactic acid. With regard to all of the microbiological parameters measured, the wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene were essentially identical. Studies of the implications of pre- and probiotics may need to be expanded to include careful analysis of their effects on the entire microbial community, rather than just a few well-known species. Further studies are needed to increase our understanding of the possible roles of currently unknown gastrointestinal bacteria in health and disease.

Animal Feed↗
PubMed2000

The lipopolysaccharide structures of Salmonella enterica serovar Typhimurium and Neisseria gonorrhoeae determine the attachment of human mannose-binding lectin to intact organisms.

Mannose-binding lectin (MBL) is an important component of the innate immune system. It binds to the arrays of sugars commonly presented by microorganisms and activates the complement system independently of antibody. Despite detailed knowledge of the stereochemical basis of MBL binding, relatively little is known about how bacterial surface structures influence binding of the lectin. Using flow cytometry, we have measured the binding of MBL to a range of mutants of Salmonella enterica serovar Typhimurium and Neisseria gonorrhoeae which differ in the structure of expressed lipopolysaccharide (LPS). For both organisms, the possession of core LPS structures led to avid binding of MBL, which was abrogated by the addition of O antigen (Salmonella serovar Typhimurium) or sialic acid (N. gonorrhoeae). Truncation of the LPS within the core led to lower levels of MBL binding. It was not possible to predict the magnitude of MBL binding from the identity of the LPS terminal sugar alone, indicating that the three-dimensional disposition of LPS molecules is probably also of importance in determining MBL attachment. These results further support the hypothesis that LPS structure is a major determinant of MBL binding.

Carbohydrate Sequence↗
PubMed1993

Mechanisms of TonB-catalyzed iron transport through the enteric bacterial cell envelope.

The recent solution of enteric bacterial porin structure, and new insights into the mechanism by which outer membrane receptor proteins recognize and internalize specific ligands, advocates the re-evaluation of TonB-dependent transport physiology. In this minireview we discuss the potential structural features of siderophore receptors and TonB, and use this analysis to evaluate both existing and new models of energy and signal transduction from the inner membrane to the outer membrane of gram-negative bacteria.

Amino Acid Sequence↗
PubMed2003

Structural and mechanistic basis of bacterial sugar nucleotide-modifying enzymes.

Recently, carbohydrates have come to the fore because of their central role in many biological processes. One area of current interest concerns the enzymatic modification of sugar nucleotides, in relation to both secondary metabolite glycosylation and the formation of complex cell surface-associated glycoconjugates. Bacteria, in particular, have proven to be a rich field in which to study these transformations, because they are often unique to specific classes of organisms. This has led to the realization that such microbial biosynthetic pathways might be exploited in the generation of novel antibiotics, or indeed serve as targets for such compounds. This work illustrates the interplay between protein structure determination, chemistry, and molecular biology in providing insight into the mechanism of such biochemical transformations.

Bacteria↗
PubMed2005

Variation in antibiosis ability, against potato pathogens, of bacterial communities recovered from the endo- and exoroots of potato crops produced under conventional versus minimum tillage systems.

The culturable component of bacterial communities found in the endoroot and associated exoroot (root zone soil) was examined in potatoes (Solanum tuberosum L.) grown under either conventional or minimum tillage systems. Bacterial species--abundance relationships were determined and in vitro antibiosis ability investigated to discover whether tillage practice or bacteria source (endo- or exoroot) influenced bacterial community structure and functional versatility. Antibiosis abilities against Phytophthora erythroseptica Pethyb. (causal agent of pink rot of potatoes), Streptomyces scabies (Thaxt.) Waksm. and Henrici) (causal agent of potato common scab), and Fusarium oxysporum Schlecht. Emend. Snyder and Hansen (causal agent of fusarium potato wilt) were selected as indicators of functional versatility. Bacterial community species richness and diversity indices were significantly greater (P = 0.001) in the exoroot than in the endoroot. While both endo- and exoroot communities possessed antibiosis ability against the phytopathogens tested, a significantly greater proportion (P = 0.0001) of the endoroot population demonstrated antibiosis ability than its exoroot counterpart against P. erythroseptica and F. oxysporum. Tillage regime had no significant influence on species-abundance relationships in the endo- or exoroot but did influence the relative antibiosis ability of bacteria in in vitro challenges against S. scabies, where bacteria sourced from minimum tillage systems were more likely to have antibiosis ability (P = 0.0151). We postulate that the difference in the frequency of isolates with antibiosis ability among endoroot versus exoroot populations points to the adaptation of endophytic bacterial communities that favour plant host defence against pathogens that attack the host systemically.

Agriculture↗
PubMed2006

Diversity of microorganisms in Fe-As-rich acid mine drainage waters of Carnoulès, France.

The acid waters (pH 2.7 to 3.4) originating from the Carnoulès mine tailings contain high concentrations of dissolved arsenic (80 to 350 mg.liter(-1)), iron (750 to 2,700 mg.liter(-1)), and sulfate (2,000 to 7,500 mg.liter(-1)). During the first 30 m of downflow in Reigous creek issuing from the mine tailings, 20 to 60% of the dissolved arsenic is removed by coprecipitation with Fe(III). The microbial communities along the creek have been characterized using terminal-restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene library analyses. The results indicate a low bacterial diversity in comparison with unpolluted water. Eighty percent of the sequences obtained are related to sequences from uncultured, newly described organisms or recently associated with acid mine drainage. As expected owing to the water chemistry, the sequences recovered are mainly related to bacteria involved in the geochemical Fe and S cycles. Among them, sequences related to uncultured TrefC4 affiliated with Gallionella ferruginea, a neutrophilic Fe-oxidizing bacterium, are dominant. The description of the bacterial community structure and its dynamics lead to a better understanding of the natural remediation processes occurring at this site.

Arsenic↗
PubMed2025

Fingolimod as a potent anti-Staphylococcus aureus: pH-dependent cell envelope damage and eradication of biofilms/persisters.

BACKGROUND: The urgent need for new antibacterial drugs has driven interest in repurposing therapies to combat Gram-positive biofilms and persisters. Fingolimod, an Food and Drug Administration (FDA)-approved drug for multiple sclerosis, shows bactericidal activity, particularly against Methicillin-resistant Staphylococcus aureus (MRSA) and biofilm-related infections. With a well-documented safety profile and strong translational potential, it aligns with World Health Organization's goals for antimicrobial repurposing. However, the action mode and mechanism of Fingolimod against gram-positive bacteria remain elusive. METHODS: This study utilized clinical Staphylococcus aureus (S. aureus), Enterococcus faecalis (E. faecalis), Streptococcus agalactiae (S. agalactiae). And their susceptibility to Fingolimod and other antibiotics was tested via Minimum Inhibitory Concentration (MIC) assays. Biofilm inhibition and hemolytic activity were evaluated using crystal violet staining, Confocal Laser Scanning Microscopy (CLSM), and hemolysis assays, respectively, while the effect of phospholipids on Fingolimod efficacy was assessed with checkerboard assays. Membrane permeability and integrity were measured using SYTOX green staining and transmission electron microscopy. Whole-genome sequencing was performed on Fingolimod-resistant S. aureus isolates to identify Single Nucleotide Polymorphisms (SNPs) linked to resistance. RESULTS: Our data indicated that Fingolimod exerted bactericidal activity against a wide spectrum of gram-positive bacteria, including S. aureus, E. faecalis, S. agalactiae. Moreover, Fingolimod could significantly eliminate the persisters, inhibit biofilm formation and eradicate in-vitro mature biofilms of S. aureus. The mechanism by which Fingolimod rapidly eradicated S. aureus involved a pH-dependent disruption of bacterial cell permeability and envelope integrity. Concomitantly, exogenous supplementation of phospholipids in the culture medium resulted in a dose-dependent increase in the MIC of Fingolimod. Specifically, the addition of 64 μg/mL of cardiolipin (CL) and phosphatidylethanolamine (PE) completely nullified the bactericidal activity of Fingolimod at a concentration of 4 times the MIC. After four months of Fingolimod exposure, the MIC values of S. aureus showed a slight increase, indicating that it is not prone to developing drug resistance. CONCLUSION: Fingolimod exhibits bactericidal activity against diverse gram-positive bacteria, with remarkable effects on S. aureus (including MRSA), disrupting bacterial cell structural integrity in a pH-dependent way and eradicating biofilms and persisters of S. aureus.

Biofilms↗
PubMed2000

Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation.

Multinucleated giant cells (MNGC) derived from avian blood monocytes present, like osteoclasts, an unusual cytoskeletal organization characterized by (1) cortical rings of actin filaments, (2) unique adhesion structures called podosomes and (3) vinculin containing focal complexes which are not visibly connected to F-actin structures. The Rho family of small GTPases plays an essential role in the regulation and organization of cellular cytoskeletal structures including F-actin and vinculin associated structures. Using bacterial toxins such as modified exoenzyme C3 (C3B) and toxin B or overexpression of constitutively active Rac and Rho proteins fused to the green fluorescent protein (GFP), we show that Rac and Rho play antagonistic roles in regulating the morphology of osteoclast-like cells. Inhibition of Rho by C3B triggered MNGC spreading whereas activated Rho promoted cell retraction. However, inhibition or activation of Rho led to complete disorganization of fibrillar actin structures, including podosomes. Toxin B inhibition of Rho, Rac and Cdc42 induced a time dependent F-actin and vinculin reorganization. Initially, actin fibers with associated adhesion plaques formed and disappeared subsequently. Finally, only small focal complexes remained at the MNGC periphery before retracting. At the time when actin fibers formed, we observed that Rac was already inhibited by toxin B. By combining C3B treatment and overexpression of a dominant negative form of Rac (N17Rac), we show that the formation of these focal adhesion and actin fiber structures required neither Rho nor Rac activity. Moreover, our results show that podosomes are extremely unstable structures since any modifications of Rho or Rac activity resulted in their dissociation.

3T3 Cells↗
PubMedDate not supplied

A three-dimensional protein model for human cytochrome P450 2D6 based on the crystal structures of P450 101, P450 102, and P450 108.

Cytochromes P450 (P450s) constitute a superfamily of phase I enzymes capable of oxidizing and reducing various substrates. P450 2D6 is a polymorphic enzyme, which is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. This deficiency leads to impaired metabolism of a variety of drugs. All drugs metabolized by P450 2D6 contain a basic nitrogen atom, and a flat hydrophobic region coplanar to the oxidation site which is either 5 or 7 A away from the basic nitrogen atom. The aim of this study was to build a three-dimensional structure for the protein and more specifically for the active site of P450 2D6 in order to determine the amino acid residues possibly responsible for binding and/ or catalytic activity. Furthermore, the structural features of the active site can be implemented into the existing small molecule substrate model, thus enhancing its predictive value with respect to possible metabolism by P450 2D6. As no crystal structures are yet available for membrane-bound P450s (such as P450 2D6), the crystal structures of bacterial (soluble) P450 101 (P450cam), P450 102 (P450BM3), and P450 108 (P450terp) have been used to build a three-dimensional model for P450 2D6 with molecular modeling techniques. Several important P450 2D6 substrates were consecutively docked into the active site of the protein model. The energy optimized positions of the substrates in the protein agreed well with the original relative positions of the substrates within the substrate model. This confirms the usefulness of small molecule models in the absence of structural protein data. Furthermore, the derived protein model indicates new leads for experimental validation and extension of the substrate model.

Amino Acid Sequence↗
PubMed1993

Reverse transcriptases from bacterial retrons require specific secondary structures at the 5'-end of the template for the cDNA priming reaction.

Multicopy single-stranded DNA (msDNA) is a peculiar molecule consisting of a single-stranded DNA that is branched out from an internal G residue of an RNA molecule (msdRNA) via a 2',5'-phosphodiester linkage. The genetic unit required for msDNA synthesis is designated "retron" and consists of msr (a gene for msdRNA), msd (a gene for msDNA), and a gene for reverse transcriptase (RT) in a single operon. To date, four different msDNAs have been isolated from Escherichia coli. They do not share any primary sequences in either RNA or DNA. To elucidate the specificity of bacterial RT for msDNA synthesis, the msr-msd region from retron-Ec67 was introduced into E. coli cells producing RT-Ec73, or the msr-msd region from retron-Ec73 into E. coli cells producing RT-Ec67. In both cases, msDNA was not synthesized. However, when the msdRNA coding regions (msr) for retron-Ec67 and -Ec73 were mutually exchanged and the chimeric genes were introduced into E. coli cells producing either RT-Ec67 or RT-Ec73, it was thus found that msDNA was produced only when msr and RT were from the same retron. Requirement of the msr region for msDNA synthesis by RT was further investigated by mutations in the msr region for retron-Ec67. These analyses revealed that there is a strict requirement for specific primary sequences as well as the secondary structure in msdRNA. This finding is discussed in relationship to the mechanism of the priming reaction of cDNA synthesis by eukaryotic retroviral RTs using tRNAs.

Bacterial Proteins↗
PubMed1969

Structure of the peptidoglycan of bacterial spores: occurrence of the lactam of muramic acid.

Six major oligosaccharides were released from the peptidoglycan of spores of Bacillus subtilis by lysozyme treatment. They were isolated and characterized as a disaccharide, tetrasaccharide, and hexasaccharide composed of equal amounts of muramic acid and glucosamine and containing two, three, and four acetyl groups, respectively. Three of the compounds were substituted by a single L-alanine residue, and the other three by a single tetrapeptide substituent on the acetylmuramic acid residue at the reducing end of each compound. The other muramic acid residue in the tetrasaccharides (and two of the three in the hexasaccharides) were shown to be present as muramic lactams, a sugar not previously found in nature and, hence, a unique spore constituent. Other features of the structure of spore peptidoglycan are discussed.

Bacillus subtilis↗
PubMedDate not supplied

[Search for alternative secondary structures of RNA, regulating expression of bacterial genes].

Expression of many bacterial genes is regulated by formation of alternative secondary RNA structure within the leader mRNA sequence. Our algorithm designed to search for these structures (basing on analysis of one nucleotide sequence) was applied to analyze operons of amino acid biosynthesis in alpha- and gamma-proteobacteria. The attenuators of these operons are predicted for genomes of some poorly known gamma-proteobacteria including Shewanella putrefaciens, attenuators of the tryptophan operon in some alpha-proteobacteria are also predicted.

Base Sequence↗
PubMed2003

Relationship between spatial and genetic distance in Agrobacterium spp. in 1 cubic centimeter of soil.

The spatial and genetic unit of bacterial population structure is the clone. Surprisingly, very little is known about the spread of a clone (spatial distance between clonally related bacteria) and the relationship between spatial distance and genetic distance, especially at very short scale (microhabitat scale), where cell division takes place. Agrobacterium spp. Biovar 1 was chosen because it is a soil bacterial taxon easy to isolate. A total of 865 microsamples 500 microm in diameter were sampled with spatial coordinates in 1 cm(3) of undisturbed soil. The 55 isolates obtained yielded 42 ribotypes, covering three genomic species based on amplified ribosomal DNA restriction analysis (ARDRA) of the intergenic spacer 16S-23S, seven of which contained two to six isolates. These clonemates (identical ARDRA patterns) could be found in the same microsample or 1 cm apart. The genetic diversity did not change with distance, indicating the same habitat variability across the cube. The mixing of ribotypes, as assessed by the spatial position of clonemates, corresponded to an overlapping of clones. Although the population probably was in a recession stage in the cube (10(3) agrobacteria g(-1)), a high genetic diversity was maintained. In two independent microsamples (500 microm in diameter) at the invasion stage, the average genetic diversity was at the same level as in the cube. Quantification of the microdiversity landscape will help to estimate the probability of encounter between bacteria under realistic natural conditions and to set appropriate sampling strategies for population genetic analysis.

Agriculture↗