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Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys166 is predicted to be the active site nucleophile of the catalytic mechanism.

A database search indicated homology between some members of the nitrilase/cyanide hydratase family, Pseudomonas aeruginosa and Rhodococcus erythropolis amidases and several other proteins, some of unknown function. BLOCK and PROFILE searches confirmed these relationships and showed that four regions of the P. aeruginosa amidase had significant homology with corresponding regions of nitrilases. A phylogenetic tree placed the P. aeruginosa and R. erythropolis amidases in a group with nitrilases but separated other amidases into three groups. The active site cysteine in nitrilases is conserved in the P. aeruginosa amidase indicating that Cys166 is the active site nucleophile.

Amidohydrolases↗

Cross-reactivity between Mycobacterium tuberculosis H37Rv and various actinomycetes and related organisms.

One hundred and forty-one strains of Actinomycetales and related organisms were investigated by immunodiffusion for the presence of antigens which cross-react with the antigens of M. tuberculosis H37Rv. The test strains comprised 86 different species names and 20 different genus names; they were mainly environmental organisms isolated from soil, plants, animals and such like. More than 90% of these strains were shown to have one or more antigens in common with the tubercle bacillus, and 77% had two or more antigens in common with this organism. Certain strains, of Nocardia and Rhodococcus, demonstrated abundant cross-reactivity with the tubercle bacilli, sharing up to 5 and 6 precipitinogens respectively.

Actinomycetales↗

Phylogeny of the Whipple's-disease-associated bacterium.

Efforts to culture and identify the intracellular bacteria associated with Whipple's disease have been unsuccessful. Nucleotide sequencing and amplification by the polymerase chain reaction was done on the bacterial 16 S ribosomal DNA present in a small-bowel biopsy specimen taken from a patient with Whipple's disease. A search by computer for similar rRNA sequences filed in databases showed the Whipple's-associated organism to be most similar to bacteria of the Rhodococcus, Streptomyces, and Arthrobacter genera, and more weakly related to mycobacteria. The biopsy specimen was estimated to contain around 10(7) cells of the organism. The probable aetiological agent for our patient's illness has not been identified previously in a patient with Whipple's disease.

Aged↗

Stereospecific microbial reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl-1H-1)-benzazepin+ ++-2-o ne.

A key intermediate, (3R-cis)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluorome thyl)- 2H-1-benzazepin-2-one (compound II or SQ32191), with high optical purity was made by the stereoselective microbial reduction of the parent ketone 1. Several strains of bacterial and yeast cultures were screened for the ability to catalyse the stereoselective reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl)-1H-1-benzazepin++ +-2,3-dione [compound I or SQ32425]. Microorganisms from the genera Nocardia, Rhodococcus, Alkaligenes, Corynebacterium, Arthrobacter, Hansenula, and Candida reduced compound I to compound II with 60-70% conversion yield. In contrast, microorganisms from the genera Pseudomonas and Acinetobacter reduced compound I stereospecifically to (trans)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromet hyl-2H- 1-benzazepin-2-one (compound III or SQ32408). Among various cultures evaluated, N. salmonicolor SC6310 effectively catalysed the transformation of compound I to compound II with 96% conversion yield at 1.5-2.0 gl-1 concentration. Compound II was isolated and identified by NMR analysis, mass spectrometry, and comparison to an authentic sample. Preparative scale fermentation process and transformation process were developed using cell suspensions of N. salmonicolor SC6310 to catalyse the transformation of compound I to compound II. The isolated compound II had a melting point of 222 degrees C (reference 221-223 degrees C), optical rotation of +130.4 (reference +128 degrees C), and optical purity of greater than 99.9% as analyzed by NMR and chiral HPLC.

Bacteria↗

Occurrence of two structural types of mercury reductases among gram-positive bacteria.

Structural variants of mercury reductase containing the N-terminal domain, which is easily cleaved by trypsin, have been found in Gram-positive bacteria with a low genomic G + C content (Bacillus, Staphylococcus and, possibly, some other genera). Mercury reductases without the N-terminal domain and relatively resistant to limited proteolysis are typical for Gram-positive bacteria with a high genomic G + C content (Arthrobacter, Citreobacterium, Micrococcus, Mycobacterium, Rhodococcus). Both types of mercury reductase genes may be located on plasmids.

Base Composition↗

Polyacrylamide gel electrophoresis analysis of ribosomal protein: a new approach for actinomycete taxonomy.

The ribosomal (r)-proteins from eleven Streptomyces strains representing various numerical taxonomic clusters were compared by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). The protein patterns were specific for each species. An attempt was made to identify one strain of Streptomyces by both traditional taxonomic methods and 2D-PAGE analysis of the r-protein patterns. Both methods identified the strain as Streptomyces lavendulae, and protein pattern analysis also showed that S. griseolavendus was a variant of S. lavendulae. Actinomycete r-protein AT-L30 exhibited electrophoretic mobility that is specific for each genus. On the basis of this observation, we analyzed AT-L30 r-proteins from numerous strains of species belonging to the genera Actinomadura, Microtetraspora, Streptosporangium, Nocardia, Rhodococcus and mycolate-less wall chemotype-IV actinomycetes. The results strongly supported the conclusions of previous work and thus proved the efficacy of r-protein analysis as a novel approach for taxonomy of actinomycetes.

Actinomycetales↗

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

Amino Acid Sequence↗

The development and evaluation of an enzyme-linked immunosorbent assay for the detection of Mycobacterium bovis.

A double antibody sandwich layer enzyme-linked immunosorbent assay (ELISA) was used to detect Mycobacterium bovis. The ELISA detected M. bovis is pure culture at concentrations of 1 x 10(5) colony-forming units (CFU) ml-1 and greater, compared to a minimum detection level of 1 x 10(6) CFU ml-1 for isolation techniques. Neither technique detected M. bovis at 1 x 10(4) CFU ml-1. The ELISA did not cross-react with common mycobacterial contaminants such as Mycobacterium avium intracellulare-scrofulaceum complex serotypes 18 and 42, M. terrae, M. fortuitum, M. flavescens and with Escherichia coli or Rhodococcus equi. Further work is needed to evaluate this assay in detecting M. bovis in tissues and the environment.

Animals↗

Analysis of the immunodominant antigens of Corynebacterium pseudotuberculosis.

Antibodies to seven antigens in a whole cell lysate of Corynebacterium pseudotuberculosis ranging in molecular mass from 22 to 120 kilodaltons (kDa) were present in sera of 40 sheep and goats infected with C. pseudotuberculosis. Three antigens of about 120, 68, and 31.5 kDa in size were consistently detected with sera from all animals and twenty-two sera had antibodies to 64, 43, 40, and 22 kDa antigens. None of these antigens were detected by sera from 160 sheep in a C. pseudotuberculosis-free research flock. An NaCl extract of C. pseudotuberculosis cells contained one major protein of about 31.5 kDa and four minor proteins of 68, 64, 43, and 22 kDa in molecular mass as shown by Coomassie Blue staining. Immunoblot analysis demonstrated that the three immunodominant antigens identified in the whole cell extract were contained in the NaCl extract. The 31.5-kDa protein was purified from the NaCl extract by fast-protein liquid chromatography gel filtration to near homogeneity. The purified 31.5-kDa protein showed phospholipase D activity as indicated by synergistic hemolysis with Rhodococcus equi factors and sphingomyelinase activity. The 31.5-kDa protein reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis. This serum also had phospholipase D neutralizing activity. On the basis of its molecular mass, biological activity, N-terminal amino acid sequence analysis, and immunoreactivity, the 31.5-kDa protein was identified as the phospholipase D exotoxin of C. pseudotuberculosis.

Amino Acid Sequence↗

Genetic differences between nitrate-negative and nitrate-positive C. pseudotuberculosis strains using restriction fragment length polymorphisms.

Corynebacterium pseudotuberculosis has been classified into two biotypes according to ability to breakdown nitrate (Biberstein et al., 1971). Restriction enzyme analysis (REA) has shown to reflect this differentiation, but numerous bands generated by this technique make interpretation difficult (Songer et al., 1988). Restriction fragment length polymorphism's (RFLP's) has become an accepted genetic tool and was used in this study to determine if differences in nitrate reduction and other phenotypic characteristics could be identified genetically. Thirteen C. pseudotuberculosis isolates from four species of domestic animals from different parts of the world were investigated for phenotypic and genetic differences. Three closely related bacteria, Corynebacterium ulcerans, Actinomyces pyogenes (previously C. pyogenes),and Rhodococcus equi (previously C. equi) were included in the study to determine if the RFLP bands were unique to C. pseudotuberculosis. All C. pseudotuberculosis isolates were positive for urease production. Some differences in maltose and sucrose fermentation ability and nitrate reduction were recorded. Genetic differences were identified between the nitrate-positive group and the nitrate-negative group using non-radioactive ribosomal RNA (rRNA) probes Southern blotted to restriction digests of ApaI, PstI, and SstI. A small number of bands were seen, with distinct differences between the nitrate-positive and the nitrate-negative strains. No genetic variations were seen between strains which reflected differences in carbohydrate fermentation. Strains isolated from different animal species and from different parts of the world could not be differentiated genetically using these three restriction enzymes.

Animals↗

Development of a serologic panel for the recognition of nocardial infections in a murine model.

A murine model was used to develop a sensitive and specific serologic test for clinical and subclinical infections caused by Nocardia. The following tests were used: (a) enzyme-linked immunosorbent assays (ELISAs) with culture filtrate and cytoplasmic extract antigens from Nocardia asteroides; (b) ELISA with N. asteroides trehalose dimycolate (cord factor); (c) indirect immunofluorescent antibody assay with whole cells of N. asteroides; and (d) Western-blot analysis for the 54 to 55-kD, 36-kD, and 62-kD proteins of N. asteroides. The sera from BALB/c mice, experimentally infected with nonlethal doses of three species of Nocardia, nine species of Mycobacterium, Rhodococcus equi, two species of Actinomadura, and two species of Streptomyces were tested using this panel. The serologic tests did, indeed, identify mice infected with nocardiae and could differentiate them from mice infected with the other actinomycetes, including mycobacteria.

Actinomycetales↗

Detection of a genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens.

The polymerase chain reaction (PCR) and automated DNA sequencing were used to detect a genetic locus, rpoB, associated with rifampin resistance in Mycobacterium tuberculosis (TB) in clinical isolates and directly in clinical specimens. Primers derived from the sequence of a TB rpoB gene fragment were used to amplify DNA from bacterial and mycobacterial isolates. An rpoB-specific PCR product was obtained for five of five TB, seven of eight other mycobacterial species, Nocardia sp., Corynebacterium sp., Streptomyces sp., Actinomyces sp., and Rhodococcus sp., but not for 15 isolates (eight genera) representing usual bacterial flora. Sequence comparison of the amplified rpoB region revealed the occurrence of TB-specific "signature nucleotides" at three positions. PCR yielded amplification products for seven of 16 clinical specimens. Five of the seven contained TB-specific DNA, as well as sequences that predicted rifampin susceptibility in accord with agar dilution results. None of ten specimens that were culture negative for TB yielded TB-specific PCR products. These results with a limited number of clinical specimens demonstrate the feasibility of direct detection by PCR of rifampin-resistant TB in clinical specimens. Such testing may serve as a rapid surrogate test for multidrug-resistant TB in laboratories with PCR and automated sequencing capability.

Base Sequence↗

Antimicrobial activity of cefotaxime tested against infrequently isolated pathogenic species (unusual pathogens).

The cefotaxime sodium spectrum of activity is very broad and includes many common species and a variety of less frequently isolated pathogens. We have reviewed the clinical microbiology literature (44 references) and the data base of the University of Iowa Hospitals and Clinics (Iowa City, IA) to collect data on the activity of cefotaxime against the less common species. Cefotaxime was consistently active against Actinobacillus actinomycetemcomitans, Capnocytophaga spp., Eikenella corrodens, Erysipelothrix rhusiopathiae, Pasteurella multocida, Plesiomonas shigelloides, and Fusobacterium nucleatum. The species Alcaligenes xylosoxidans, Flavobacterium spp., Stenotrophomonas (Xanthomonas) maltophilia, Bacillus cereus, Listeria monocytogenes, and Rhodococcus equi were uniformly cefotaxime resistant. For many other species there was considerable variation in reported minimum inhibitory concentrations. These data may be helpful in guiding therapy of unusual infections, particularly in the case of fastidious species, where the appropriate susceptibility testing methodology may not be immediately or routinely available.

Bacteria, Anaerobic↗

Discovering hidden candidate plastic-degrading enzymes: Combined multi-omics and machine learning strategy.

Plastic pollution poses a major threat to the stability of natural ecosystems as well as human health. Microbial enzymes have long been considered a potential resource for targeted biodegradation but, except for a few successful cases, the discovery of efficient enzymes has proved challenging. Aiming to accelerate the process, we propose an approach combining metagenomics, metatranscriptomics and semi-supervised learning that selects promising plastic-degrading candidate enzymes from the proteome of relevant microorganisms. Tested on a dataset of over 10,000 microbial proteins, ranking models consistently prioritize known plastic-degrading enzymes, achieving an area under the cumulative distribution function curve above 0.96, with leave-one-family-out cross-validation indicating that performance is largely retained across protein families. As a case study, this work focuses on mixed microbial cultures exposed for extended periods to polyethylene, polyethylene terephthalate, and polyurethane substrates. The prevalent species after selective enrichment were functionally characterized, finding Rhodococcus aetherivorans as the most relevant species in two of the five cultures under investigation. Among the top-ranked proteins, several have high structural similarity with known enzymes despite not being identified by sequence similarity search. Moreover, according to metatranscriptomics results, several of these enzymes were found to be expressed at the same level or above that of annotated enzymes, suggesting that they may have functional relevance. Overall, this work highlights the potential of integrating multi-omics with data-driven methods for enzyme discovery and for accelerating the development of biotechnological solutions to plastic pollution.

Biodegradation, Environmental↗

Effect of gas velocity and influent concentration on biofiltration of gasoline off-gas from soil vapor extraction.

This study was conducted to evaluate the effects of gas inlet concentration and velocity on the biofiltration of gasoline vapor. Gasoline vapor was treated using a compost biofilter operated in an upflow mode for about 3 months. The inlet concentration of gasoline total petroleum hydrocarbon (TPH) ranged from about 300 to 7000 mgm(-3) and gas was injected at velocities of 6 and 15 mh(-1) (empty bed residence time (EBRT)=10 and 4 min, respectively). The maximum elimination capacities of TPH at 6 and 15 mh(-1) found in this research were over 24 and 19 gm(-3) of filling material h(-1), respectively. TPH removal data was fit using a first-order kinetic relationship. In the low concentration range of 300-3000 mg m(-3), the first-order kinetic constants varied between about 0.10 and 0.29 min(-1) regardless of gas velocities. At TPH concentrations greater than 3000 mgm(-3), the first-order kinetic constants were about 0.09 and 0.07 min(-1) at gas velocities of 6 mh(-1) and 15 mh(-1), respectively. To evaluate microbial dynamics, dehydrogenase activity, CO2 generation and microbial species diversity were analyzed. Dehydrogenase activity could be used as an indicator of microbial activity. TPH removal corresponded well with CO2 evolution. The average CO2 recovery efficiency for the entire biofilter ranged between 60% and 70%. When the gas velocity was 6 mh(-1), most of the microbial activity and TPH removal occurred in the first quarter of the biofilter. However, when the gas velocity was 15 mh(-1), the entire column contributed to removal. Spatial and temporal variations in the biofilter microbial population were also observed. Nearly 60% of the colonies isolated from the compost media prior to biofiltration were Bacillus. After 90 days of biofiltration, the predominant species in the lower portion (0-50 cm) of the filter were Rhodococcus, while Pseudomonas and Acinetobacter dominated the upper portion (75-100 cm).

Bacteria↗

Isolation and characterization of a diverse group of phenylacetic acid degrading microorganisms from pristine soil.

A diverse range of microorganisms capable of growth on phenylacetic acid as the sole source of carbon and energy were isolated from soil. Sixty six different isolates were identified and grouped according to 16S rRNA gene RFLP analysis. Subsequent sequencing of 16S rDNA from selected strains allowed further characterization of the phenylacetic acid degrading population isolated from soil. Nearly half (30) of the isolates are Bacillus species while the rest of the isolates are strains from a variety of genera namely, Arthrobacter, Pseudomonas, Rhodococcus, Acinetobacter, Enterobacter, Flavobacterium, and Paenibacillus. Sixty-one of the sixty-six strains reproducibly grew in defined minimal liquid culture medium (E2). All strains isolated grew when at least one hydroxylated derivative of phenylacetic acid was supplied as the carbon source, while 59 out of the 61 strains tested, accumulated ortho-hydroxyphenylacetic acid in the assay buffer; when pulsed with phenylacetic acid. Oxygen consumption experiments failed to indicate a clear link between phenylacetic acid and hydroxy-substituted phenylacetic acid in isolates from a broad range of genera.

Bacteria↗

Pet ownership in immunocompromised children--a review of the literature and survey of existing guidelines.

Pet ownership has been associated with both emotional and physical health benefits. However, owning pets may also pose health risks to immunocompromised patients through zoonotic transmission of disease. Our initial impression was that there is a lack of any evidence base in information given by health care professionals regarding these risks. We therefore aimed to produce evidence-based guidelines addressing this issue. A Pubmed search was undertaken and a variety of literature on zoonoses reviewed. Existing guidelines were evaluated and a survey of all Paediatric Oncology Centres in the UK performed. There is a paucity of level 1 and 2 data addressing this issue and clearly more studies, particularly Randomised Controlled Trials (RCTs), are required. Nevertheless, general themes emerged and certain specific guidance was produced based on that produced by the Centres for Disease Control and Prevention in the US. Animal-associated pathogens of concern include Toxoplasma gondii, Cryptosporidium spp., Salmonella spp., Campylobacter spp., Giardia lamblia, Rhodococcus equi, Bartonella spp., Bordetella bronchiseptica, Chlamydia psittaci and dermatophytes. Despite this, the literature would suggest that with the exception of Bartonella henselae and dermatophytes only a relatively small number of infections in people are likely to be associated with pet contact. The majority of pet species do not appear to pose a major risk to immunocompromised children. Some animals, particularly reptiles, should be avoided because of the high risk of salmonellosis. Guidelines include general advice on good hygiene practices, veterinary care, pet foods, purchasing of new pets and age restrictions. Health care professionals should actively enquire about household pets and provide accurate information and practical advice on how to minimise the risk of infection. However, the overall benefits of the human-animal bond must be considered and with proper handling and husbandry immunocompromised patients should be able to continue to enjoy the significant benefits of pet ownership.

Age Factors↗

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase from Bacillus laterosporus J1, is a member of the alpha/beta-hydrolase fold superfamily.

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase (GCA) isolated from Bacillus laterosporus J1, has been conventionally grouped as the only member of class V GCA, although its amino acid sequence shares less than 10% identity with members of other classes of GCA. Instead, it shows higher sequence similarities with Rhodococcus sp. strain MB1 cocaine esterase (RhCocE) and Acetobacter turbidans alpha-amino acid ester hydrolase (AtAEH), members of the alpha/beta-hydrolase fold superfamily. Homology modeling and secondary structure prediction indicate that the N-terminal region of J1 acylase has an alpha/beta-hydrolase folding pattern. The catalytic triads in RhCocE and AtAEH were identified in J1 acylase as S125, D264 and H309. Mutations to alanine at these positions were found to completely inactivate the enzyme. These results suggest that J1 acylase is a member of the alpha/beta-hydrolase fold superfamily with a serine-histidine-aspartate catalytic triad.

Amidohydrolases↗