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PubMed1998

Expression of maize gamma zein C-terminus in Escherichia coli.

Prolamins containing a highly conserved cysteine-rich C-terminal domain have been poorly expressed as soluble protein in model systems such as Escherichia coli. Possible reasons have included a combination of the reducing environment of the bacterial cytoplasm and protein secondary structure. Using a bacterial thioredoxin fusion expression system, full-length native gamma zein, native gamma zein C-terminus, and modified gamma zein C-terminus, containing 13 amino acid changes, were found to accumulate up to 58, 50, and 42% of the total cellular protein, respectively. The native gamma zein C-terminus fusion protein was six times more soluble (70%) than the full-length fusion protein (12%), four times more soluble than the N-terminus (19%), and eight times more soluble than the modified C-terminus (9%). The modified C-terminal domain contained amino acid changes that improved the lysine, isoleucine, and tryptophan content, while removing two evolutionarily conserved cysteines and one nonconserved cysteine. Expression of the native C-terminal domain without thioredoxin resulted in decreased solubility (13%) and decreased expression (8%). In contrast, coexpression with thioredoxin resulted in a sevenfold increase in solubility (86%). These results suggest that insolubility of full-length gamma zein results from structural interactions of the N-terminus and that solubility of the C-terminal domain is dependent on proper disulfide bond formation. The ability to express the C-terminal domain of gamma zein as soluble protein should allow future identification of important structural elements in gamma zein and similar proteins.

Amino Acid Sequence↗
PubMed2005

Nitrobacter and Nitrospira genera as representatives of nitrite-oxidizing bacteria: detection, quantification and growth along the lower Seine River (France).

Pollution from agriculture and urban effluents influences the ecology and biochemical functioning of the Seine River. Nitrification dominates nitrogen transformations downstream of the effluents of the Paris wastewater treatment plant (WWTP) at Achères, treating, by activated sludge the wastewater of 6.5 million inhabitant equivalents from Paris and its suburbs, without nitrification and denitrification treatment. It discharges effluents containing large amounts of nitrogen, ammonium mostly (approximately 30 mg L(-1) N-NH(4+) L(-1)), on average 45 mg L(-1) of suspended particulate matter, high quantities of total organic carbon (approximately 30 mg C L(-1)) largely biodegradable (40%), and high concentration in total phosphorus ( approximately 3 mg Tot P L(-1)), as well as microorganisms. Ammonium, brought into the river system, is slowly nitrified in the lower Seine River and especially in the freshwater estuary. The nitrifying activities can be observed by measuring inorganic nitrogen compound concentrations and potential activities. To understand the contributions of the WWTP effluents, the upstream agricultural runoff water and the Seine tributaries, it is useful to investigate the bacterial community. Whereas ammonia oxidation has been widely studied, the second step, i.e. nitrite oxidation, is less well understood. We have previously analysed the ammonium-oxidizing bacterial (AOB) community in the Seine (Cébron, A., Berthe, T., Garnier, J., 2003. Nitrification and nitrifying bacteria in the lower Seine River and estuary (France). Appl. Environ. Microbiol. 69, 7091-7100; Cébron, A., Coci, M., Garnier, J., Laanbroek, H.J., 2004. DGGE analysis of the ammonia oxidizing bacterial community structure in the lower Seine River: impact of the Paris wastewater effluents. Appl. Environ. Microbiol. 70, 6726-6737), and focus here on the composition of the nitrite-oxidizing bacterial (NOB) community. As no general molecular probe targeting all known NOBs is currently available, we chose to target and quantify (by competitive PCR) the two genera Nitrobacter and Nitrospira assumed to be the major players in nitrite oxidation in freshwater environments. Nitrobacter species were dominant in the upstream Seine River basin but Nitrospira was the dominant NOB downstream of the WWTP. These two genera were equally represented in WWTP effluents. In the Seine River estuary, especially in the salinity gradient, the Nitrobacter proportion increases and that of Nitrospira disappears, possibly due dilution by seawater.

Bacteria↗
PubMed2002

Substrate routes to the buried active site may vary among cytochromes P450: mutagenesis of the F-G region in P450 2B1.

Until recently, all known structures of bacterial cytochromes P450 suggested that substrate access to the buried active site occurred via the F-G region, a surface loop distal to the heme cavity. However, the structure of P450 51 indicates a large opening from the protein surface along the I helix N-terminus, at right angles to the F-G channel. The single available microsomal P450 structure (2C5) does not obviously favor one potential access route over the other. To determine whether the F-G region forms part of the substrate access channel in the microsomal cytochrome P450 2B1, 11 residues between positions 208 and 230 were substituted with smaller and larger side chains in a highly expressed truncated form of the enzyme. Steady-state kinetic parameters were determined with the substrates testosterone, 7-ethoxy-4-trifluoromethylcoumarin (7-EFC), and 7-benzyoxyresorufin (7-BR). The largest changes, 2-6-fold increases in k(cat) with testosterone and 7-EFC, were observed for L209A, which also exhibits an altered testosterone metabolite profile and probably forms part of the active site roof. F219W demonstrated little or no activity with any of the three substrates examined, although the K(s) value for benzphetamine binding was unaltered. S221F showed little activity with 7-BR. No significant changes were observed in K(m)(testosterone) or S(50)(7-EFC) values for any of the mutants, in stark contrast to the 10-fold and 100-fold changes in K(m) observed for mutants in this region of other cytochromes P450. The minimal changes in 2B1 do not support access via the F-G region of 2B1 and suggest the alternate access route identified in P450 51.

Base Sequence↗
PubMed1996

[Lipopolysaccharides containing sialic acid].

Sialic acids which are important constituents of animal tissue glycoconjugates are also present in antigens of some bacterial strains. Capsular polysaccharides with sialic acid have been extensively studied whereas little is known on lipopolysaccharides which contain sialic acid. The paper presents review of the data concerning structure of bacterial endotoxic lipopolysaccharides. Methodological peculiarities were described. Specially emphasized were endotoxins of Escherichia coli O24, O56, O104, Salmonella toucra O48, Citrobacter freundii O37 and Hafnia alvei strain PCM2386.

Bacterial Toxins↗
PubMed1989

The physiological role of eubacterial histone-like proteins.

This minireview reflects the change in views on the role of histone-like proteins which has occurred in the 1980 s. Initially these proteins were regarded as analogous to eukaryotic histones though distinguished from the latter by much lower strength of binding to DNA. This was attributed to the greater dynamics of the structure of bacterial chromatin. The accumulation in recent years of results testifying to the absence of protein HU in central region of the nucleoid and the participation of this protein in almost all processes involving the recognition of specific DNA sequences by regulatory proteins forces the rejection of the concept of "histone-likeness" and favors the concept of a general, non-specific enhancer of the recognition of sequences acting by causing changes in the secondary structure of a given DNA region.

Bacterial Proteins↗
PubMed1993

Ultrastructure of the surface film of bacterial colonies.

The structure of the surface of colonies of various Gram-negative and Gram-positive bacteria was examined by transmission electron microscopy. The results indicate that bacterial colonies in the course of their development produce a film which becomes thicker with increased duration of growth. The basic part of the film is an elementary membrane, which is a stable structure with a large surface area. The inner and outer surfaces of the film membrane are covered by amorphous layers. These layers are thicker in the surface film of Gram-negative bacterial colonies than in those of Gram-positive bacteria. Membrane vesicles from the bacterial colonies take part in the formation of the surface film. The presence of the film on the surface of the colonies of different bacteria suggests that this structure may play an important role.

Brevibacterium↗
PubMed2000

Correlating a protein structure with function of a bacterial mechanosensitive channel.

MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes. First isolated from Escherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension. Recently the structure of a functionally uncharacterized MscL homologue from Mycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 A. Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E. coli membranes and azolectin proteoliposomes. Furthermore, we show that M. tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization. Finally, we apply functional clues acquired from the E. coli MscL to the M. tuberculosis channel and show a mechanistic correlation between these channels. However, the inability of the M. tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E. coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.

Bacterial Proteins↗
PubMed1995

Structural organization and assembly of flagellar hook protein from Salmonella typhimurium.

The terminal regions of monomeric hook protein from Salmonella typhimurium are known to be highly mobile and exposed to the solvent. Although hook protein exhibits an unusual far-UV circular dichroism spectrum, resembling that of random coil structures, our calorimetric experiments clearly demonstrate that the molecule has a compact ordered core. The compact part probably consists of three domains as suggested by deconvolution analysis of the calorimetric melting profiles. Secondary structure prediction, together with the analysis of far-UV circular dichroism spectra, has shown that the domains of monomeric hook protein contain beta-sheeted structures without significant alpha-helical content. The polymerization of hook protein is accompanied by the stabilization of its disordered terminal regions into a predominantly alpha-helical domain. Evaluation of circular dichroism data suggests that about 45 terminal residues are involved in helical segments. Coiled-coil prediction indicates that whereas the whole carboxy-terminal helical region of hook protein has a strong bundle-forming potential, there is only a single short amino-terminal segment exhibiting weak coiled-coil forming tendencies. The formation of alpha-helical bundles is commonly believed to be a key event during the polymerization of the axial structure of bacterial flagella. To clarify the role of helical bundle formation in hook assembly, proteolytic fragments of hook protein with truncations of various lengths in their carboxy-terminal disordered regions were generated, and their polymerization behavior was investigated. We found that even fragments completely lacking the main helix-forming carboxy-terminal regions can polymerize into filaments in vitro under appropriately high salt concentrations. Our results suggest that, although helical bundle formation may occur during self-assembly, governing precise subunit packing and playing an important role in the stabilization of hook filaments, it is not the principal interaction mainly responsible for the development of their filamentous structure.

Bacterial Proteins↗
PubMed1994

The three-dimensional structures of mutants of porphobilinogen deaminase: toward an understanding of the structural basis of acute intermittent porphyria.

Mutations in the human gene for the enzyme porphobilinogen deaminase give rise to an inherited disease of heme biosynthesis, acute intermittent porphyria. Knowledge of the 3-dimensional structure of human porphobilinogen deaminase, based on the structure of the bacterial enzyme, allows correlation of structure with gene organization and leads to an understanding of the relationship between mutations in the gene, structural and functional changes of the enzyme, and the symptoms of the disease. Most mutations occur in exons 10 and 12, often changing amino acids in the active site. Several of these are shown to be involved in binding the primer or substrate; none modifies Asp 84, which is essential for catalytic activity.

Amino Acid Sequence↗
PubMed2005

The solution structure of the Josephin domain of ataxin-3: structural determinants for molecular recognition.

The Josephin domain plays an important role in the cellular functions of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We have determined the solution structure of Josephin and shown that it belongs to the family of papain-like cysteine proteases, sharing the highest degree of structural similarity with bacterial staphopain. A currently unique structural feature of Josephin is a flexible helical hairpin formed by a 32-residue insertion, which could determine substrate specificity. By using the Josephin structure and the availability of NMR chemical shift assignments, we have mapped the enzyme active site by using the typical cysteine protease inhibitors, transepoxysuccinyl-L-eucylamido-4-guanidino-butane (E-64) and [L-3-trans-(propylcarbamyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline (CA-074). We also demonstrate that the specific interaction of Josephin with the ubiquitin-like domain of the ubiquitin- and proteasome-binding factor HHR23B involves complementary exposed hydrophobic surfaces. The structural similarity with other deubiquitinating enzymes suggests a model for the proteolytic enzymatic activity of ataxin-3.

Ataxin-3↗
PubMed1978

Translational specificity of Bacillus stearothermophilus ribosomes.

The translational specificity of Bacillus stearothermophilus ribosomes was studied by determining the effectiveness of various synthetic RNAs as templates at 37 degrees and at higher temperatures. The effectiveness of poly(G,U) was maximal at a G:U ratio of 1:3; it declined with lower G content because of reduced ribosomal affinity for the RNA and, with higher G, because of interference by secondary structure. The effectiveness of poly(A,C,G,U) also declined when secondary structure was increased by increasing (G+C) content. Escherichia coli ribosomes exhibited a similar specificity for poly-(G,U), but had a lower sensitivity to interference by RNA secondary structure. In both bacterial species, sensitivity to secondary structure was determined by the 30S ribosomal subunit.

Base Sequence↗
PubMed2003

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel.

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.

Amino Acid Sequence↗
PubMed2005

Adherence of Streptococcus suis to porcine endothelial cells.

Streptococcus suis can cause invasive diseases in pigs and humans, such as meningitis or arthritis. Adherence to and invasion of endothelial cells might represent important steps in survival and spread of S. suis within the host. We tested in vitro adherence and invasion of S. suis strains using a porcine brain microvascular and aortal endothelial cell line. Four S. suis strains were tested with and without prior treatment with porcine serum containing anti-S. suis antibodies. Strains included a capsular serotype 2 strain and its non-encapsulated isogenic mutant strain, as well as two non-typeable (NT) strains, which expressed no capsule under our experimental conditions. Strains adhered to both cell lines to different extents depending on encapsulation and pre-treatment with porcine immune serum. The serotype 2 strain showed almost no adherence, whereas the non-encapsulated mutant strain adhered strongly. Similarly, both NT strains adhered substantially better than the serotype 2 strain. Pre-treatment of bacteria with porcine serum increased adherence of the encapsulated serotype 2 strain and decreased adherence of the non-encapsulated strains. None of the strains was able to efficiently invade either of the two cell lines, except for one NT strain, which showed a very low extend of invasion. Our results suggest that S. suis can adhere to but not invade porcine endothelial cells, and that this interaction may involve different bacterial surface structures, such as capsular polysaccharides and/or binding sites for serum components.

Animals↗
PubMed2001

Porphyromonas gingivalis fimbriae inhibit caspase-3-mediated apoptosis of monocytic THP-1 cells under growth factor deprivation via extracellular signal-regulated kinase-dependent expression of p21 Cip/WAF1.

Apoptotic regulation of monocytes/macrophages appears to be closely associated with chronic inflammatory reactions. Since it was demonstrated earlier that certain bacterial cell components are involved in apoptotic regulation of these cells, in the present study, we investigated whether the bacterial fimbria, an important cell structure involved in bacterial adherence to host cells, regulates apoptosis of human monocytic THP-1 cells induced under growth factor deprivation. To investigate this point, we used fimbriae of Porphyromonas gingivalis, a pathogen causing periodontal disease, which is a chronic inflammatory disease. The fimbriae inhibited apoptosis of the cells under growth factor deprivation. This inhibitory action of the fimbriae was completely neutralized by anti-fimbrial antibody. The fimbriae stimulated activation of extracellular signal-regulated kinase (ERK) and expression of cyclin-dependent kinase inhibitor p21 Cip/WAF1 (p21) in the cells. The stimulatory effect of the fimbriae on the expression of the p21 protein was inhibited by treatment with PD98059, a specific inhibitor of ERK. The cell apoptosis was inhibited by treatment with Ac-DEVD-CHO, an inhibitor of caspase-3. The fimbriae inhibited the serum withdrawal-induced cleavage of the caspase-3 proform and poly(ADP-ribose) polymerase, one of the caspase-3 substrates. Furthermore, PD98059 and antisense p21 oligonucleotide blocked the fimbrial inhibition of apoptosis and caspase-3 activation of the cells induced by serum withdrawal. These results show that the bacterial fimbriae inhibited apoptosis of THP-1 cells induced under growth factor deprivation via ERK-dependent expression of p21. The present study suggests that bacterial fimbriae act as potent regulators of chronic inflammatory disease, e.g., periodontal disease, through blocking apoptosis of monocytes/macrophages.

Apoptosis↗
PubMed2002

Molecular structure and physiological function of chloride channels.

Cl- channels reside both in the plasma membrane and in intracellular organelles. Their functions range from ion homeostasis to cell volume regulation, transepithelial transport, and regulation of electrical excitability. Their physiological roles are impressively illustrated by various inherited diseases and knock-out mouse models. Thus the loss of distinct Cl- channels leads to an impairment of transepithelial transport in cystic fibrosis and Bartter's syndrome, to increased muscle excitability in myotonia congenita, to reduced endosomal acidification and impaired endocytosis in Dent's disease, and to impaired extracellular acidification by osteoclasts and osteopetrosis. The disruption of several Cl- channels in mice results in blindness. Several classes of Cl- channels have not yet been identified at the molecular level. Three molecularly distinct Cl- channel families (CLC, CFTR, and ligand-gated GABA and glycine receptors) are well established. Mutagenesis and functional studies have yielded considerable insights into their structure and function. Recently, the detailed structure of bacterial CLC proteins was determined by X-ray analysis of three-dimensional crystals. Nonetheless, they are less well understood than cation channels and show remarkably different biophysical and structural properties. Other gene families (CLIC or CLCA) were also reported to encode Cl- channels but are less well characterized. This review focuses on molecularly identified Cl- channels and their physiological roles.

Animals↗
PubMed2002

De novo ligand design with explicit water molecules: an application to bacterial neuraminidase.

Most computer-aided drug design methods ignore the presence of crystallographically-determined water molecules in the binding site of a target protein. In this paper, our de novo ligand design methods are applied to the X-ray crystal structure of bacterial neuraminidase in the presence of some selected water molecules. We have found that, for this particular protein, the complete removal of all bound water molecules leads to difficulties in generating any potential ligands if the unsatisfied hydrogen-bonding sitepoints left by removing these water molecules are to be satisfied by a ligand. As more of the crystallographically determined water molecules are allowed in the binding site, it becomes much easier to generate ligands in larger numbers and with wider chemical diversity. This example shows that, in some cases, bound water molecules can be more accessible for hydrogen bonding to an incoming ligand than the actual protein binding sitepoints associated with them. From the point of view of de novo ligand design, water molecules can thus act as versatile amphiprotic hydrogen-bonding sitepoints and reduce the conformational constraints of a particular binding site.

Bacteria↗
PubMed2005

Soil bacterial diversity responses to root colonization by an ectomycorrhizal fungus are not root-growth-dependent.

The hypothesis tested in this present study was that the ectomycorrhizosphere effect on the bacterial community was not root-growth-dependent. The impacts of ectomycorrhizal infection (Pisolithus albus COI007) and a chemical fertilization to reproduce the fungal effect on root growth were examined on (1) the structure of bacterial community and (2) fluorescent pseudomonad and actinomycete populations in the mycorrhizosphere of Acacia auriculiformis using both culture-independent and culture-dependent methods. A. auriculiformis plants were grown in disinfested soil in pots with or without addition of the ectomycorrhizal fungus or N/P/K fertilization (to reproduce the fungal effect on root growth) for 4 months and then transferred to 20-L pots filled with nondisinfested sandy soil. The fungal and fertilizer applications significantly improved the plant growth after 4-month culture in the disinfested soil. In the nondisinfested cultural substrate, these positive effects on plant growth were maintained. The total soil microbiota was significantly different within the treatments as revealed from DNA analysis [denaturing gradient gel electrophoresis (DGGE)]. The structure of fluorescent pseudomonad populations was also affected by fungal and fertilizer applications. In contrast, no qualitative effect was observed for the actinomycete communities within each treatment, but fungal inoculation significantly decreased the number of actinomycetes compared to the fertilizer application treatment. These results show that the mycorrhizosphere effect is not root-growth-dependent but is mainly due to the presence of the ectomycorrhizal fungus and more particularly to the extramatrical mycelium.

Acacia↗