Proteomic analysis of the benzoate degradation pathway in Acinetobacter sp. KS-1

The purpose of this study was to perform proteome analysis of Acinetobacter sp. KS-1, a bacterium capable of degrading benzoate as a sole carbon source. In order to understand the benzoate degradation pathway used by strain KS-1, proteomes of benzoate-cultured and succinate-cultured KS-1 were comparatively analyzed by two dimensional gel electrophoresis (2-DE). Eighteen protein spots proteins were exclusively induced from the benzoate-cultured strain KS-1. Of these 18 spots, two benzoate-degrading enzymes (catechol 1,2-dioxygenase and beta-ketoadipate succinyl-CoA transferase) were identified by MS/MS analysis by MALDI-TOF/TOF mass spectrometry, which suggests that strain KS-1 degrades benzoate by the beta-ketoadipate pathway. DEAE-chromatography suggested that strain KS-1 induced only one type of catechol 1,2-dioxygenase during benzoate degradation. The catechol 1,2-dioxygenase was purified using three steps of ammonium sulfate precipitation, DEAE-sepharose, and Mono-Q chromatography. The purified catechol 1,2-dioxygenase of strain KS-1 had strong dioxygenase activity for 4-methylcatechol as well as catechol. Sequencing analysis using N-terminal and internal amino acid sequences showed that this catechol 1,2-dioxygenase is highly homologous with catechol 1,2-dioxygenase of Acinetobacter radioresistens. These results suggest that comparative proteomic analysis of biodegrading bacteria cultured under different conditions may be a useful initial step toward the elucidation of the aromatic compound degradation pathway.     

Beta-1,2-mannosylation of Candida albicans mannoproteins and glycolipids differs with growth temperature and serotype

Increasing the growth temperature from 28 to 37 degrees C reduced the expression of beta-1,2-oligomannoside epitopes on mannoproteins of Candida albicans serotypes A and B. In contrast, beta-1,2-mannosylation of phospholipomannan (PLM) remained constant despite a slight decrease in the relative molecular weight (M(r)) of this compound. At all growth temperatures investigated, serotype A PLM displayed an M(r) and an antigenicity different from those of serotype B PLM when they were tested with a panel of monoclonal antibodies.     

Potential Immunogenic Polypeptides of Burkholderia pseudomallei Identified by Shotgun Expression Library and Evaluation of Their Efficacy for Serodiagnosis of Melioidosis

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.     

Occurrence and properties of glutathione S-transferases in phenol-degrading Pseudomonas strains

Pseudomonas sp. strains, able to degrade aromatic compounds such as phenol, were chosen to investigate the occurrence and characteristics of glutathione S-transferases (GSTs). Affinity chromatography purification showed the presence of at least one GST in each studied strain. The purified proteins exhibited a great variety in the N-terminal sequences and different enzyme activities with the standard GST substrates tested. Two Pseudomonas strains, M1 and CF600, were chosen to investigate the GST activities under different growth conditions. Therefore, cells were grown either on phenol or on different nonaromatic carbon sources in the presence and absence of increasing phenol concentrations. In strain M1 a strong correlation between the activities of the catechol 1,2-dioxygenase and GST was observed in all the tested conditions. Moreover, growth on different organic acids also affected GST activity levels, with a negative correlation with the specific growth rate determined by each substrate. These results suggest a possible function of GST as a response to specific metabolic conditions determined by phenol toxicity and/or catabolism and the metabolic status of the cells. The same experiments performed with the CF600 strain did not show induction of GST activity in any of the tested conditions, indicating that GST_CF600 probably has a different role in cell metabolism. Native gel electrophoresis gave indications that GST dimerization could be an important process in the modulation of GST activity.     

Anaerobic benzene biodegradation--a new era

Benzene is biodegraded in the absence of oxygen under a variety of terminal electron-accepting conditions. However, the mechanism by which anaerobic benzene degradation occurs is unclear. Phenol and benzoate have been consistently detected as intermediates of anaerobic benzene degradation, suggesting that the hydroxylation of benzene to phenol is one of the initial steps in anaerobic benzene degradation. The conversion of phenol to benzoate could then occur by the carboxylation of phenol to form 4-hydroxybenzoate followed by the reductive removal of the hydroxyl group to form benzoate. 13C-Labeling studies suggest that the carboxyl carbon of benzoate is derived from one of the carbons of benzene. Although the fumarate addition reaction is commonly used to activate many hydrocarbons for anaerobic degradation, the large activation energy required to remove hydrogen from the benzene ring argues against such an approach for anaerobic benzene metabolism. The alkylation of benzene to toluene has been detected in several mammalian tissues, and offers an interesting alternate hypothesis for anaerobic benzene degradation in microbial systems. In support of this, anaerobic benzene degradation by Dechloromonas strain RCB, the only known species to degrade benzene in the absence of oxygen, is stimulated by the addition of vitamin B12 and inhibited by the addition of propyl iodide which is consistent with the involvement of a corrinoid enzymatic step. Alkylation of benzene to toluene is also consistent with labeling data that suggests that the carboxyl carbon of benzoate is derived from one of the benzene carbons. However, it is difficult to envision how phenol would be formed if benzene is alkylated to toluene. As such, it is possible that diverse mechanisms for anaerobic benzene degradation may be operative in different anaerobic microorganisms.     

Strain A/J mouse lung adenoma growth patterns vary when induced by different carcinogens.

The histogenesis of mouse lung adenomas is currently being investigated in several laboratories. Based upon studies of a limited number of carcinogens in different mouse strains, some investigators suggest that all lung adenomas in mice are derived from alveolar type II cells, whereas others suggest a Clara cell origin for a majority of the tumors. This report differs from previous investigations in that 12 different carcinogens were evaluated for the types of lung tumor growth patterns they induced in a single mouse strain (strain A mice). The carcinogens aflatoxin B1 (AFB1), benzo(a)pyrene (BP), 1,2-dimethylhydrazine (DMH), 3-methylcholanthrene (MCA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosomethylurea (MNU) induced tumors with a predominantly solid/alveolar growth pattern, whereas N-nitrosodiethylamine (NDEA) induced predominantly papillary tumors. Most of the other carcinogens induced a higher proportion of lung tumors with the solid/alveolar growth pattern than with the papillary growth pattern; however, ratios between the 2 growth patterns varied. If, as suggested by others, solid tumors are derived from alveolar type II cells and papillary tumors from Clara cells, then carcinogens may differ with respect to their ability to transform one cell type or the other.     

Critical steps in degradation of chloroaromatics by rhodococci I. Initial enzyme reactions involved in catabolism of aniline,phenol and benzoate by Rhodococcus sp. An 117 and An 213

Two bacterial isolates (i.e. strains An 117 and An 213) capable of growing with aniline, phenol as well as benzoate as the sole carbon and energy source were studied with respect to (i) their taxonomic position, (ii) the enzyme reactions which initiate catabolism of the respective aromatic compounds, and (iii) the general type of regulation of the respective enzymes. Both isolates were established to be representatives of the actinomycete-genus Rhodococcus. Experiments with resting cells and cell-free extracts, respectively, revealed that in the two strains under study catabolism of each of the unsubstituted aromatic compounds occurs via the β-ketoadipate pathway (with catechol as the central metabolite) due to the action of inducible enzymes. Although being potent inducers of the ring-cleaving catechol 1,2-dioxygenase in strains An 117 and An 213, all of the monochlorinated derivatives of aniline, phenol and benzoate, respectively, failed to support cell growth of the organisms. Cis, cis-muconic acid proved to be non-metabolizable by resting An 117 and An 213 cells, although substantial (inducible) muconate cycloisomerase activity was detectable in crude extracts prepared from the respective cell preparations. NADPH-depending phenol hydroxylase activity could be demonstrated in crude extracts from phenol-grown An 117 and An 213 cells. Evidence is presented that in both Rhodococcus strains under study substantial de novo synthesis of at least the initial aromatics-oxygenating enzymes can be induced by phenol and aniline, respectively, even in the presence of either succinate (An 117) or acetate (An 213) which are known to be ortho-pathway catabolites.     

Genetic heterogeneity of G and F protein genes from Argentinean human metapneumovirus strains

Human metapneumovirus (hMPV) is a newly identified paramixovirus, associated with respiratory illnesses in all age groups. Two genetic groups of hMPV have been described. The nucleotide sequences of the G and F genes from 11 Argentinean hMPV strains (1998-2003) were determined by RT-PCR and direct sequencing. Phylogenetic analysis showed that hMPV strains clustered into two main genetic lineages, A and B. Strains clustered into A group were split into two sublineages, A1 and A2. All strains belonging to group B clustered with representative strains from sublineage B1. No Argentinean strains belonged to sublineage B2. F sequences showed high percentage identities at nucleotide and amino acid levels. In contrast, G sequences showed high diversity between A and B groups. Most changes observed in the deduced G protein sequence were amino acid substitutions in the extracellular domain, and changes in stop codon usage leading to different lengths in the G proteins. High content of serine and threonine residues were also shown, suggesting that this protein would be highly glycosylated. The potential sites for N- and O-glycosylation seem to have a different conservation pattern between the two main groups. This is the first report on the genetic variability of the G and F protein genes of hMPV strains in South America. Two main genetic groups and at least three subgroups were revealed among Argentinean hMPV strains. The F protein seems to be highly conserved, whereas the G protein showed extensive diversity between groups A and B.     

Key enzymes for the degradation of benzoate,m- and p-hydroxybenzoate by some members of the order Actinomycetales

A preliminary screening of numerous species of the order Actinomycetales, especially of the genera Mycobacterium, Nocardia, Rhodococcus, Pseudonocardia, and Streptomyces, showed that many of them are able to metabolize benzoate (B) and p-hydroxybenzoate (pHB) as indicated by growth and change of color of the pH-indicator of an agar medium. Subsequent experiments with liquid cultures which allowed the analysis of substrate utilization by thin layer chromatography confirmed these results. The study of the degradative pathway proved that B was metabolized via catechol (C), pHB via protocatechuate (P) and m-hydroxybenzoate (mHB) via gentisate (G). The aromatic ring of C and P was subjected to an ortho-cleavage; only one strain of Noc. asteroides degraded C via a meta-cleavage, but P via an ortho-cleavage. Cell free extracts of four selected organisms exhibited activity of C-1,2-dioxygenase (C-1,2-O) and/or P-3,4-dioxygenase (P-3,4-O), depending on the growth substrate used for precultivation. In Streptomyces C-1,2-O was only found in cells grown on B, and P-3,4-O only in cells grown on pHB. On the contrary, in Rhodococcus rhodochrous B-cells oxidized C as well as P, while P-cells possessed only P-3,4-O-activity.     

Influenza type B neuraminidase can replace the function of type A neuraminidase

Influenza A and B viruses do not form reassortants with each other, presumably due to selection at either the RNA or protein level. Although differences in the promoter sequences of type A and B viruses have been studied, selection at the protein level has not been addressed. In this paper we describe experiments to determine whether differences in structure and/or function of the neuraminidase (NA) protein preclude formation of A/B NA reassortants. Influenza type A (N9) NA or B/Lee/40 NA expressed from plasmids can support multicycle growth of a NA-deficient type A virus (NWS-Mvi), indicating that their function in tissue culture is similar. To determine whether the type A or B NA supplied in trans can be incorporated into the virion of NWS-Mvi, the virus grown in NA-expressing cells was purified by sucrose gradient centrifugation. In each case there was a peak of NA activity coincident with the virus peak, indicating that some NA protein is packaged into the virion. The experiments suggest that, in spite of large sequence differences, the functions of the head, stalk, signal-anchor, and cytoplasmic domains of type A and B NAs are similar in tissue culture. Thus, lack of formation of A/B NA reassortant viruses is not due to restriction at the protein level.     

Role of RAV-0 genes in the permissive replication of subgroup E avian leukosis viruses on line 15Bev1 CEF

Rous associated virus type-0 (RAV-0) is a replication-competent endogenous virus of chickens which grows more efficiently on chick embryo fibroblasts (CEFs) from line 15B chickens than on CEFs from line K28. Differences in viral growth on these two sources of cells have been attributed to an early event in the retrovirus life-cycle, at or before viral DNA synthesis. Five in vitro constructed avian leukosis viruses (ALVs) as well as RAV-0 (a subgroup E ALV), RAV-1 (subgroup A), and RAV-2 (subgroup B) have been assessed for their relative growth on 15Bev1 and K28 CEFs. More efficient replication on 15Bev1 CEFs than on K28 CEFs was determined by subgroup E-encoding sequences in env. Subgroup A and B envelope sequences as well as viral LTR, gag, and pol sequences did not obviously bias relative rates of viral replication on the two cell types. We suggest that the unusually permissive replication of subgroup E viruses on 15B CEFs is a receptor-mediated phenomenon and that the line 15B receptor for subgroup E ALVs is more efficient than that of line K28.     

Biodegradation of phenylbenzoate and some of its derivatives by Scedosporium apiospermum.

Scedosporium apiospermum, a recently isolated phenol-degrading hyphomycete, was shown to be able to productively utilise the diaryl ester phenylbenzoate as its sole source of carbon and energy. The characterisation of degradation intermediates together with the detection of the corresponding catabolic enzymes in crude extracts enabled us to propose a pathway for the degradation of this diaryl ester. According to our results, an inducible esterase initiated the biodegradation of phenylbenzoate by hydrolysing the ester bond to yield stoichiometric amounts of phenol and benzoate. While phenol was catabolised via catechol and hydroxyhydroquinone, the benzoate was further degraded via the protocatechuate branch of the ortho-pathway. In addition, the fungus utilised p-tolylbenzoate and 4-chlorophenylbenzoate by employing similar catabolic sequences.     

Effect of oxygen radical scavengers on K-cell cytolysis.

We have shown that the hydroxyl radical scavengers sodium benzoate, phenol, dimethyl sulfoxide, sodium formate, and mannitol protect chicken erythrocyte (Ec) target cells from lysis by K-cells in plaque assays. The protection afforded by benzoate, phenol, mannitol, and dimethyl sulfoxide was abrogated if the target cells were pretreated with sodium chromate. Neither superoxide dismutase nor catalase protected Ec target cells, indicating that superoxide and H2O2 are not involved in lysis. The lysis obtained on chromium-treated Ec target cells in the presence of benzoate, phenol, and mannitol is likely due to singlet oxygen, because 1,4-diazabicyclo[2.2.2]octane and bilirubin, singlet oxygen scavengers, protected chromium-treated Ec target cells in the presence of phenol. Ec target cells not treated with chromium were not protected by either of the singlet oxygen scavengers.     

F7 and type 1-like fimbriae from three Escherichia coli strains isolated from urinary tract infections: protein chemical and immunological aspects.

Fimbriae from three Escherichia coli strains, C1212, C1214, and C1023, isolated from urinary tract infections, have been purified and characterized by determination of the N-terminal sequences, amino acid composition, and molecular weights of their respective subunits. Furthermore, their immunological interrelationships have been investigated. The three strains all harbored more than one fimbrial species each. Immunologically different type 1-like fimbriae, termed 1A, 1B, and 1C, with highly homologous N-terminal sequences were isolated, of which strain C1212 possessed 1A and 1C, strain C1214 possessed 1A and 1B, and strain C1023 possessed 1A and 1C. Type 1A is known to cause a mannose-sensitive hemagglutination similar to that described for type 1 fimbriae, whereas the functions of types 1B and 1C are not yet known. Strain C1212, in addition, harbored the F7 fimbrial antigen which causes mannose-resistant hemagglutination and adherence to urinary epithelial cells. The N-terminal structure of this antigen seems to indicate a possible evolutionary kinship to the type 1-like fimbriae, although they are immunologically unrelated. Our results indicate that fimbriation of pathogenic wild-type strains can be of an intricate variety.     

A single-step DNA extraction procedure for the detection of serum hepatitis B virus sequences by the polymerase chain reaction.

A rapid single-step procedure for the isolation of low molecular weight DNA using guanidinium thiocyanate and phenol as protein denaturants is described and applied for the detection of specific hepatitis B virus (HBV) DNA sequences from serum samples by the polymerase chain reaction (PCR). The novel technique is efficient and, when compared to the standard proteinase K/phenol/chloroform method has the advantage of being faster and easily adaptable to the routine processing of a high number of clinical samples by PCR and spot hybridization techniques.     

Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins.

A set of vectors possessing the genes for aspartate semialdehyde dehydrogenase (asd) and the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B) has been developed. These vectors allow operon or gene fusions of foreign gene epitopes at the C-terminal end of LT-B. Two groups of vectors have been constructed with and without leader sequences to facilitate placing of the foreign antigen in different cell compartments. Two Streptococcus sobrinus genes coding for principal colonization factors, surface protein antigen A (SpaA), and dextranase (Dex), have been fused into the 3' end of the LT-B gene. Resulting protein fusions of approximately 120 to 130 kDa are extremely well recognized by antibodies directed against both SpaA and Dex as well as against LT-B domains and retain the enzymatic activity of dextranase and the biological activity of LT-B in that they bind to GM1 gangliosides. Maximum antigenicity was obtained with the vector possessing an intervening linker of at least six amino acids with two proline residues. Some of the fusion proteins also exhibited another property of LT-B in that they were exported into the periplasm where they oligomerized. LT-B-SpaA and LT-B-Dex hybrid proteins are expressed stably and at a high level in avirulent Salmonella typhimurium vaccine strains which are being used to investigate their immunogenicity and types of induced immune responses. The fusion vectors will also be useful for production and purification of LT-B fusion antigens to be used and evaluated in other vaccine compositions.     

Aromatic-dependent Salmonella as live vaccine presenters of foreign epitopes as inserts in flagellin.

Synthetic oligonucleotides specifying amino acid sequences identified as epitopes of various foreign antigens (cholera toxin subunit B, hepatitis B surface protein and others) have been inserted at an EcoRV-EcoRV deletion site in a cloned Salmonella flagellin gene; the resulting plasmids, when placed in flagellin-negative Escherichia coli or Salmonella sp. strains, caused production of flagellin expressing the epitope. If the chimeric flagellin allowed formation of flagella, the epitope was exposed at the surface of the flagellar filaments. A delta aroA flagellin-negative S. dublin live vaccine strain given plasmids carrying various chimeric flagellin genes was administered to mice, etc. Serum antibody specific for the foreign epitope was in all cases evoked by parenteral administration; oral route administration was effective in the case of two epitopes of hepatitis B surface protein but not effective for several other epitopes. Several i.p. inocula of the live vaccine strain with an insert corresponding to the 15 N-terminal amino acids of the M protein of Streptococcus pyogenes type 5 evoked M-specific antibody with opsonic activity, and the mice were (incompletely) protected against a lethal challenge of S. pyogenes type 5. The non-virulence of Salmonella sp. strains with complete blocks in the aromatic biosynthesis pathway, even for animals with genetically determined or other defects in host defences, can be completely accounted for by their requirement for p-aminobenzoic acid, since non-leaky pabB mutations caused similar attenuation. Two transposon insertions at aroE caused little or no attenuation, presumably because they did not result in complete block of the relevant step in biosynthesis. The limited growth of delta aroA strains in mouse tissues parallels that which precedes the bacteriostasis caused by addition of a sulphonamide to a growing broth culture of a sulphonamide-sensitive strain; the final cessation of growth in each case presumably results from inability to initiate new protein chains with a formyl-methionine unit when the original folic acid content of the bacteria has been diluted out by residual growth.     

Control of Aβ release from human neurons by differentiation status and RET signaling

Few studies have compared the processing of endogenous human amyloid precursor protein (APP) in younger and older neurons. Here, we characterized LUHMES cells as a human model to study Alzheimer's disease-related processes during neuronal maturation and aging. Differentiated LUHMES expressed and spontaneously processed APP via the secretase pathways, and they secreted amyloid β (Aβ) peptide. This was inhibited by cholesterol depletion or secretase inhibition, but not by block of tau phosphorylation. In vitro aged cells increased Aβ secretion without upregulation of APP or secretases. We identified the medium constituent glial cell line-derived neurotrophic factor (GDNF) as responsible for this effect. GDNF-triggered Aβ release was associated with rapid upregulation of the GDNF coreceptor “rearranged during transfection” (RET). Other direct (neurturin) or indirect (nerve growth factor) RET activators also increased Aβ, whereas different neurotrophins were ineffective. Downstream of RET, we found activation of protein kinase B (AKT) to be involved. Accordingly, inhibitors of the AKT regulator phosphatidylinositol-3-kinase completely blocked GDNF-triggered AKT phosphorylation and Aβ increase. This suggests that RET signaling affects Aβ release from aging neurons.