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PubMed2005

An optimized system for expression and purification of secreted bacterial proteins.

In this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-c-myc eptiope fusion proteins. Without regard to their initial solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, purified under denaturing conditions by immobilized metal affinity chromatography, and refolded by rapid dilution into a solution containing only Tris buffer and sodium chloride. Following concentration on the same resin under native conditions, each protein was eluted for further purification and/or characterization. Preliminary studies on a test set of 12 secreted proteins ranging in size from 13 to 130 kDa yielded between 10 and 50 mg of fusion protein per liter of induced culture at greater than 90% purity, as judged by Coomassie-stained SDS-PAGE. Of the nine proteins further purified, analytical gel filtration chromatography indicated that each was a monomer in solution and circular dichroism spectroscopy revealed that each had adopted a well-defined secondary structure. While there are many potential applications for this system, the results presented here suggest that it will be particularly useful for investigators employing structural approaches to understand protein function, as attested to by the crystal structures of three proteins purified using this methodology (B.V. Geisbrecht, B.Y. Hamaoka, B. Perman, A. Zemla, D.J. Leahy, J. Biol. Chem. 280 (2005) 17243-17250).

Amino Acid Sequence↗
PubMed2006

Effect of bacterial protein meal on protein and energy metabolism in growing chickens.

This experiment investigates the effect of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism, and carcass chemical composition of growing chickens. Seventy-two Ross male chickens were allocated to four diets, each in three replicates with 0% (D0), 2% (D2), 4% (D4), and 6% BPM (D6), BPM providing up to 20% of total dietary N. Five balance experiments were conducted when the chickens were 3-7, 10-14, 17-21, 23-27, and 30-34 days old. During the same periods, 22-h respiration experiments (indirect calorimetry) were performed with groups of 6 chickens (period 1), 5 chickens (period 2), and one chicken (periods 3-5). After each balance period, one chicken in each cage was killed and the carcass weight was recorded. Chemical analyses were performed on the carcasses from periods 1, 3, and 5. Weight gain, feed intake, and feed conversion rate were found to be similar for all diets. Chickens on D0 retained 1.59 g N x kg(-0.75) x d(-1), significantly more than chickens on D2, D4, and D6, which retained 1.44 g, 1.52 g, and 1.50 g N x kg(-0.75) x d(-1), respectively. This was probably caused by the higher nitrogen content of DO. Neither the HE (p = 0.92) nor the retention of energy (p = 0.88) were affected by diet. Carcass composition was similar between diets, in line with the values for protein and energy retention found in the balance and respiration experiments. It was concluded that the overall protein and energy metabolism as well as carcass composition were not influenced by a dietary content of up to 6% BPM corresponding to 20% of dietary N.

Animal Feed↗
PubMed1977

The firA gene, a locus involved in the expression of rifampicin resistance in Escherichia coli. II. Characterisation of bacterial proteins coded by lambdafirA transducing phages.

The firA gene probably codes for an essential component of the transcription machinery in E. coli. Bacterial proteins coded by lambdafirA transducing phages have been examined after infection of a UV-irradiated lambda lysogen, and 2 major fir-specific proteins have been characterized. The larger protein has a molecular weight of 27,000 daltons. The smaller protein, of 17,000 daltons, is produced in a considerable molar excess over the larger protein, is basic and binds strongly to DNA-, to DEAE- and to phospho-cellulose. This protein is clearly visible upon 2-dimensional gel electrophoresis of unfractionated E. coli protein, showing that it is present in the cell in large quantities. Evidence is presented to suggest that this protein may be identical to the Kappa factor of Schäfer and Zillig (1973).

Bacterial Proteins↗
PubMed1993

Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase lose their ability to enter cultured cells. In addition, a short-lived capacity to enter cells may be important during infection so that bacterial invasiveness is limited to certain times and host sites during pathogenesis.

Cells, Cultured↗
PubMed1989

Ig-binding bacterial proteins also bind proteinase inhibitors.

Protein G is a streptococcal cell wall protein with separate binding sites for IgG and human serum albumin (HSA). In the present work it was demonstrated that alpha 2-macroglobulin (alpha 2M) and kininogen, two proteinase inhibitors of human plasma, bound to protein G, whereas 23 other human proteins showed no affinity. alpha 2M was found to interact with the IgG-binding domains of protein G, and in excess alpha 2M inhibited IgG binding and vice versa. A synthetic peptide, corresponding to one of the homologous IgG-binding domains of protein G, blocked binding of protein G to alpha 2M. Protein G showed affinity for both native and proteinase complexed alpha 2M but did not bind to the reduced form of alpha 2M, or to the C-terminal domain of the protein known to interact with alpha 2M receptors on macrophages. Binding of protein G to alpha 2M and kininogen did not interfere with their inhibitory activity on proteinases, and the interaction between protein G and the two proteinase inhibitors was not due to proteolytic activity of protein G. The finding that protein G has affinity for proteinase inhibitors was generalized to comprise also other Ig binding bacterial proteins. Thus, alpha 2M and kininogen, were shown to bind both protein A of Staphylococcus aureus and protein L of Peptococcus magnus. The results described above suggest that Ig-binding proteins are involved in proteolytic events, which adds a new and perhaps functional aspect to these molecules.

Bacterial Proteins↗
PubMed1999

Following the leader: bacterial protein export through the Sec pathway.

Significant strides have been made during the past 20 years in our understanding of protein secretion across the bacterial inner membrane. Specialized chaperones select secretory polypeptide chains and usher them to a membrane-embedded preprotein translocase. This unique molecular machine envelops the polymeric substrate and migrates along its length in defined, energy-dependent steps. Consequently, preproteins are gradually pumped into the periplasm where they acquire their native, folded conformation.

Adenosine Triphosphatases↗
PubMed2005

Nitrogen and energy balance in growing mink (Mustela vison) fed different levels of bacterial protein meal produced with natural gas.

The objective of this study was to estimate the effect of increasing the dietary content of bacterial protein meal (BPM) on energy and protein metabolism in growing mink kits. Sixteen male mink kits of the standard brown genotype were randomly fed one of four diets: A control (Diet I) based on high-quality fish meal, and three experimental diets in which 20% (Diet II), 40% (Diet III) and 60% (Diet IV) of the digested nitrogen (DN) was replaced with BPM. Nitrogen balance and respiration experiments (indirect calorimetry) were carried out when the animals were approximately 9.5, 14.5, 17.5, 23.5 and 28.5 weeks of age. The apparent digestibility of crude protein and energy decreased significantly with increasing dietary BPM. The retained nitrogen was 0.45, 0.54, 0.52 and 0.40 g/kg0.75 on Diets I, II, III and IV, respectively, the observed differences between diets being non-significant (p = 0.06). Heat production (HE) was between 645 and 665 kJ/kg0.75 on all diets (p = 0.78). Retained energy (RE) was approximately 150-160 kJ/kg0.75 on Diets I to III, whereas it was -11 kJ/kg0.75 on Diet IV, the differences being significant (p< 0.001). A lower feed intake and apparent digestibility of energy caused the negative RE on Diet IV. The amount of HE from oxidation of protein decreased from 32.7% on Diet I to 26.6% on Diet IV, and oxidation of fat increased from 53.8% on Diet I to 63.5% Diet IV. In conclusion, protein and energy metabolism remained unaffected when up to 40% of DN was derived from BPM.

Animal Feed↗
PubMed1985

[Protein and amino acid metabolism in the digestive tract of growing young bulls. 2. Feed protein flow into the duodenum determined with 2,6-diaminopimelic acid as a marker for crude bacterial protein].

The experimental ascertainment of the pure feed protein flowing into the duodenum on the basis of the calculation of the difference between the NH3-free crude protein flowing into the duodenum and the bacterial crude protein determined by means of DAPA showed the following results after the testing of 28 different rations at a dry matter intake adequate to the production level: With a variation of the pure protein in the crude protein content of the ration between 40 and 90%, the quota of feed protein flowing into the duodenum is 52.5%, and with a variation of the pure protein in the crude protein content between 80 and 90% it is 40.6%. The quota of feed protein flowing into the duodenum shows a negative correlation to the apparent digestibility of the organic matter (y=231.7 -2.52x +/- 14.5). With a DM-intake adequate to the production level the quota of feed protein flowing into the duodenum is neither influenced by the flow rate (kg digesta/kg DM-intake) nor by the 'dilution rate' (g bacteria-free DMD/kg live weight 0.75/h).

Amino Acids↗
PubMedDate not supplied

Incorporation of N from intravenously administered 15N labelled urea into the bacterial protein in the sheep.

The experiment carried out on two wethers demonstrated that nitrogen of intravenously injected urea, labelled with 15N was incorporated into total and bacterial nitrogen fraction of the digesta flowing through the rumen and duodenum. The amount of 15N in the bacterial fraction flowing throught the rumen and duodenum was relatively low in comparison with the amount of 15N in the total nitrogen (14,8% and 8,1% in the rumen and 6,6% and 7,9% in the duodenum. The ratio of the amount of bacterial-N to total-N in the rumen content (12,7 and 7,5%) was only slightly lower than the ratio of bacterial 15N to total 15N. In the duodenum this ratio was a little higher (8,7 and 10,0%). Blood urea nitrogen was utilized only partly in biosynthesis of bacterial protein. The results showed that only a small amount of blood urea nitrogen retained in the organism was utilized for microbial protein synthesis and the majority in some different way.

Animals↗
PubMed2006

Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi.

We applied an immunoscreening technique, in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed during human infection with Salmonella enterica serovar Typhi, the cause of typhoid fever. We were able to assign a functional classification to 25 of 35 proteins identified by IVIAT. Of these 25, the majority represent proteins with known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in fimbrial structure and biogenesis, antimicrobial resistance, heavy metal transport, bacterial adhesion, and extracytoplasmic substrate trafficking as well as secreted hydrolases. The 10 remaining antigens represent proteins with unknown functions. Of the 35 identified antigens, four had no immunoreactivity when probed with control sera from individuals never exposed to serovar Typhi organisms; these four included PagC, TcfB, and two antigens of unknown function encoded by STY0860 and STY3683. PagC is a virulence factor known to be upregulated in vivo in S. enterica serovar Typhimurium infection of mice. TcfB is the major structural subunit of a fimbrial operon found in serovar Typhi with no homolog in serovar Typhimurium organisms. By examining differential immunoreactivities in acute- versus convalescent-phase human serum samples, we found specific anti-PagC and anti-TcfB immunoglobulin G responses in patients with serovar Typhi bacteremia. Serovar Typhi antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and they may have diagnostic, therapeutic, or preventive uses.

Antigens, Bacterial↗
PubMed1991

Antibiotic GE2270 a: a novel inhibitor of bacterial protein synthesis. II. Structure elucidation.

GE2270 A, produced by Planobispora rosea ATCC 53773, inhibits Gram-positive bacteria and anaerobes by acting on the bacterial protein synthesis. The structure has been determined by physico-chemical methods applied to the intact molecule and to the main hydrolysis products. Characterization by UV, IR, NMR (double quantum filter COSY), acid-base ionization, elemental analysis and FAB-MS indicated that GE2270 A is a highly modified peptide having MW 1,289 and formula C56H55N15O10S6, and a weak basic function, and that it belongs to the thiazolyl peptide group of antibiotics. Acid hydrolysis yielded a main product (MW 634), responsible for the chromophoric absorption, and a number of hydrolyzed products of lower MW. 13C NMR inverse techniques and MS studies (EI, positive ion chemical ionization, and collision induced dissociation FAB-MS-MS experiments) on GE2270 A, the chromophoric compound, and the other hydrolysis products led to the complete identification of the various amino acid residues and their sequence. Two out of the six chiral centers have been determined. The structure is thought to originate from modification of a chain of 14 amino acids in a process which creates 6 thiazole rings and one pyridine. The modification process also closes the linear polypeptide to form a cyclic part with an attached side-chain. GE2270 A plausibly has a similar biosynthetic origin to that of other thiazolyl peptide antibiotics such as nosiheptide and micrococcin.

Actinomycetales↗
PubMed2005

Prediction of functional class of novel bacterial proteins without the use of sequence similarity by a statistical learning method.

A substantial percentage of the putative protein-encoding open reading frames (ORFs) in bacterial genomes have no homolog of known function, and their function cannot be confidently assigned on the basis of sequence similarity. Methods not based on sequence similarity are needed and being developed. One method, SVMProt (http://jing.cz3.nus.edu.sg/cgi-bin/svmprot.cgi), predicts protein functional family irrespective of sequence similarity (Nucleic Acids Res. 2003;31:3692-3697). While it has been tested on a large number of proteins, its capability for non-homologous proteins has so far been evaluated for a relatively small number of proteins, and additional tests are needed to more fully assess SVMProt. In this work, 90 novel bacterial proteins (non-homologous to known proteins) are used to evaluate the capability of SVMProt. These proteins are such that none of their homologs are in the Swiss-Prot database, their functions not clearly described in the literature, and they themselves and their homologs are not included in the training sets of SVMProt. They represent proteins whose function cannot be confidently predicted by sequence similarity methods at present. The predicted functional class of 76.7% of each of these proteins shows various levels of consistency with the literature-described function, compared to the overall accuracy of 87% for the SVMProt functional class assignment of 34,582 proteins that have at least one homolog of known function. Our study suggests that SVMProt is capable of assigning functional class for novel bacterial proteins at a level not too much lower than that of sequence alignment methods for homologous proteins.

Artificial Intelligence↗
PubMed1995

Antibiotic GE37468 A: a new inhibitor of bacterial protein synthesis. I. Isolation and characterization.

GE37468 A is a new thiazolyl peptide antibiotic obtained by fermentation of Streptomyces sp. strain ATCC 55365. It inhibits bacterial protein synthesis by acting on elongation factor Tu and is structurally and functionally related to the GE2270 class of EF-Tu inhibitors. It is active in vitro against Gram-positive bacteria and Bacteroides fragilis, and protects mice against Staphylococcus aureus infection.

Animals↗
PubMed1998

Are bacterial proteins part of the matrix of kidney stones?

An extracellular protein, produced from Pseudomonas fluorescens strain D with molecular mass of 41.5 kDa was partially purified. Its first 12 amino acid sequence shows strong similarity to a sequence reported to belong to a protein isolated from a urate-calcium oxalate stone (Binnette & Binnette, Scan Microsc1994; 2: 233-239). A possible involvement of bacterial proteins in stone matrix is discussed.

Amino Acid Sequence↗
PubMed1994

Comparison of systemic and mucosal priming for mucosal immune responses to a bacterial protein antigen given with or coupled to cholera toxin (CT) B subunit, and effects of pre-existing anti-CT immunity.

Intraperitoneal immunization with a bacterial protein antigen conjugated to cholera toxin B subunit (CTB) was generally less effective than intragastric or intranasal immunization in generating mucosal IgA antibodies, and in priming the mucosal immune system to respond to intragastric or intranasal boosting. Previous intragastric or intranasal immunization which generated high levels of mucosal and circulating antibodies to CTB did not suppress mucosal IgA responses induced by intragastric or intranasal immunization with bacterial antigen conjugated to or mixed with CTB, but serum antibody responses were inhibited depending on the route of immunization and whether CTB was conjugated to or mixed with the antigen.

Administration, Intranasal↗
PubMed1988

Development of a highly sensitive bead-ELISA to detect bacterial protein toxins.

A highly sensitive sandwich enzyme-linked immunosorbent assay to detect bacterial toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The entire assay could be completed within 3.5 hr. The sensitivity of this bead-ELISA was found to be quite high with various bacterial toxins: less than 20 pg/ml for thermostable direct hemolysin of Vibrio parahaemolyticus, less than 60 pg/ml for Shiga toxin, less than 20 pg/ml for VT2 (Shiga-like toxin II) of Escherichia coli, less than 200 pg/ml for heat-labile enterotoxin of E. coli, and less than 6 pg/ml for cholera enterotoxin.

Bacterial Proteins↗
PubMed1992

Stimulation of Candida albicans transition by human chorionic gonadotrophin and a bacterial protein.

Candida albicans, a dimorphic fungus, is involved commonly in human infections with the mycelium form more associated with pathogenicity. The influence of various hormones and a bacterial protein on the transition from blastospore to mycelium was assessed. Human luteinizing hormone (hLH), chorionic gonadotrophin (hCG), and an hCG-like material purified from a bacteria, Xanthomonas maltophilia (PCG), were able to increase the rate of transition when compared with the controls. The effect of the two hormones and the bacterial peptide were specific, as human follicle stimulating hormone (hFSH), thyroid stimulating hormone (hTSH), growth hormone (hGH), prolactin (hPrl) and rat and bovine LH (rLH, bLH), and bovine albumin and gamma globulin did not affect the transition. The binding of 125I-hCG or 125I-LH to spheroplasts of Candida albicans were competitively displaced by hCG, hLH, and PCG. Scatchard analysis of binding of all three ligands revealed two binding sites with a high-affinity nM Kd. Thus, hCG, hLH, and PCG induce transition of Candida albicans from a blastospore state to a mycelium form, suggesting that these hormones may modify the pathogenicity of Candida albicans.

Bacterial Proteins↗