CYPminer: an automated cytochrome P450 identification, classification, and data analysis tool for genome data sets across kingdoms.

BACKGROUND: Cytochrome P450 monooxygenases (termed CYPs or P450s) are hemoproteins ubiquitously found across all kingdoms, playing a central role in intracellular metabolism, especially in metabolism of drugs and xenobiotics. The explosive growth of genome sequencing brings a new set of challenges and issues for researchers, such as a systematic investigation of CYPs across all kingdoms in terms of identification, classification, and pan-CYPome analyses. Such investigation requires an automated tool that can handle an enormous amount of sequencing data in a timely manner. RESULTS: CYPminer was developed in the Python language to facilitate rapid, comprehensive analysis of CYPs from genomes of all kingdoms. CYPminer consists of two procedures i) to generate the Genome-CYP Matrix (GCM) that lists all occurrences of CYPs across the genomes, and ii) to perform analyses and visualization of the GCM, including pan-CYPomes (pan- and core-CYPome), CYP co-occurrence networks, CYP clouds, and genome clustering data. The performance of CYPminer was evaluated with three datasets from fungal and bacterial genome sequences. CONCLUSIONS: CYPminer completes CYP analyses for large-scale genomes from all kingdoms, which allows systematic genome annotation and comparative insights for CYPs. CYPminer also can be extended and adapted easily for broader usage.

ß-Lactam resistance development affects binding of penicillin-binding proteins (PBPs) of Clostridium perfringens to the fluorescent penicillin, BOCILLIN FL.

Alteration in the binding of bacterial penicillin-binding proteins (PBPs) to ß-lactams is important in the development of drug resistance. The PBPs of wild type Clostridium perfringens ATCC 13124 and three ß-lactam-resistant mutants were compared for the ability to bind to a fluorescent penicillin, BOCILLIN FL. The binding of the high molecular weight protein PBP1, a transpeptidase, to BOCILLIN FL was reduced in all of the resistant strains. In contrast, the binding of BOCILLIN FL to a low molecular weight protein, PBP6, a D-alanyl-d-alanine carboxypeptidase that was more abundant in all three resistant strains, was substantially increased. A competition assay with ß-lactams reduced the binding of all of the PBPs, including PBP6, to BOCILLIN FL. ß-Lactams enhanced transcription of the putative gene for PBP6 in both wild type and resistant strains. This is the first report showing that mutations in a high molecular weight PBP and overexpression of a low molecular weight PBP in resistant C. perfringens strains affected their binding to ß-lactams.

Bacterial Populations Associated with Smokeless Tobacco Products.

There are an estimated 8 million users of smokeless tobacco products (STPs) in the United States, and yet limited data on microbial populations within these products exist. To better understand the potential microbiological risks associated with STP use, a study was conducted to provide a baseline microbiological profile of STPs. A total of 90 samples, representing 15 common STPs, were purchased in metropolitan areas in Little Rock, AR, and Washington, DC, in November 2012, March 2013, and July 2013. Bacterial populations were evaluated using culture, pyrosequencing, and denaturing gradient gel electrophoresis (DGGE). Moist-snuff products exhibited higher levels of bacteria (average of 1.05 × 106 CFU/g STP) and diversity of bacterial populations than snus (average of 8.33 × 101 CFU/g STP) and some chewing tobacco products (average of 2.54 × 105 CFU/g STP). The most common species identified by culturing were Bacillus pumilus, B. licheniformis, B. safensis, and B. subtilis, followed by members of the genera Oceanobacillus, Staphylococcus, and Tetragenococcus. Pyrosequencing analyses of the 16S rRNA genes identified the genera Tetragenococcus, Carnobacterium, Lactobacillus, Geobacillus, Bacillus, and Staphylococcus as the predominant taxa. Several species identified are of possible concern due to their potential to cause opportunistic infections and reported abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N'-nitrosamines. This report provides a microbiological baseline to help fill knowledge gaps associated with microbiological risks of STPs and to inform potential regulations regarding manufacture and testing of STPs. IMPORTANCE: It is estimated that there 8 million users of smokeless tobacco products (STPs) in the United States; however, there are limited data on microbial populations that exist within these products. The current study was undertaken to better understand the potential microbiological risks associated with STP use and provide a baseline microbiological profile of STPs. Several bacterial species were identified that are of possible concern due to their potential to cause opportunistic infections. In addition, some species have abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N'-nitrosamines. Overall, this report provides a microbiological baseline to help fill knowledge gaps related to the microbiological risks of STPs and to inform potential regulations regarding the manufacture and testing of STPs.

Comparative functional pan-genome analyses to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon metabolism in the genus Mycobacterium.

BACKGROUND: The bacterial genus Mycobacterium is of great interest in the medical and biotechnological fields. Despite a flood of genome sequencing and functional genomics data, significant gaps in knowledge between genome and phenome seriously hinder efforts toward the treatment of mycobacterial diseases and practical biotechnological applications. In this study, we propose the use of systematic, comparative functional pan-genomic analysis to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon (PAH) metabolism in the genus Mycobacterium. RESULTS: Phylogenetic, phenotypic, and genomic information for 27 completely genome-sequenced mycobacteria was systematically integrated to reconstruct a mycobacterial phenotype network (MPN) with a pan-genomic concept at a network level. In the MPN, mycobacterial phenotypes show typical scale-free relationships. PAH degradation is an isolated phenotype with the lowest connection degree, consistent with phylogenetic and environmental isolation of PAH degraders. A series of functional pan-genomic analyses provide conserved and unique types of genomic evidence for strong epistatic and pleiotropic impacts on evolutionary trajectories of the PAH-degrading phenotype. Under strong natural selection, the detailed gene gain/loss patterns from horizontal gene transfer (HGT)/deletion events hypothesize a plausible evolutionary path, an epistasis-based birth and pleiotropy-dependent death, for PAH metabolism in the genus Mycobacterium. This study generated a practical mycobacterial compendium of phenotypic and genomic changes, focusing on the PAH-degrading phenotype, with a pan-genomic perspective of the evolutionary events and the environmental challenges. CONCLUSIONS: Our findings suggest that when selection acts on PAH metabolism, only a small fraction of possible trajectories is likely to be observed, owing mainly to a combination of the ambiguous phenotypic effects of PAHs and the corresponding pleiotropy- and epistasis-dependent evolutionary adaptation. Evolutionary constraints on the selection of trajectories, like those seen in PAH-degrading phenotypes, are likely to apply to the evolution of other phenotypes in the genus Mycobacterium.

Dynamic Response of Mycobacterium vanbaalenii PYR-1 to BP Deepwater Horizon Crude Oil.

We investigated the response of the hydrocarbon-degrading Mycobacterium vanbaalenii PYR-1 to crude oil from the BP Deepwater Horizon (DWH) spill, using substrate depletion, genomic, and proteome analyses. M. vanbaalenii PYR-1 cultures were incubated with BP DWH crude oil, and proteomes and degradation of alkanes and polycyclic aromatic hydrocarbons (PAHs) were analyzed at four time points over 30 days. Gas chromatography-mass spectrometry (GC-MS) analysis showed a chain length-dependent pattern of alkane degradation, with C12 and C13 being degraded at the highest rate, although alkanes up to C28 were degraded. Whereas phenanthrene and pyrene were completely degraded, a significantly smaller amount of fluoranthene was degraded. Proteome analysis identified 3,948 proteins, with 876 and 1,859 proteins up- and downregulated, respectively. We observed dynamic changes in protein expression during BP crude oil incubation, including transcriptional factors and transporters potentially involved in adaptation to crude oil. The proteome also provided a molecular basis for the metabolism of the aliphatic and aromatic hydrocarbon components in the BP DWH crude oil, which included upregulation of AlkB alkane hydroxylase and an expression pattern of PAH-metabolizing enzymes different from those in previous proteome expression studies of strain PYR-1 incubated with pure or mixed PAHs, particularly the ring-hydroxylating oxygenase (RHO) responsible for the initial oxidation of aromatic hydrocarbons. Based on these results, a comprehensive cellular response of M. vanbaalenii PYR-1 to BP crude oil was proposed. This study increases our fundamental understanding of the impact of crude oil on the cellular response of bacteria and provides data needed for development of practical bioremediation applications.

Biotransformations of organic compounds mediated by cultures of Aspergillus niger.

Many different organic compounds may be converted by microbial biotransformation to high-value products for the chemical and pharmaceutical industries. This review summarizes the use of strains of Aspergillus niger, a well-known filamentous fungus used in numerous biotechnological processes, for biochemical transformations of organic compounds. The substrates transformed include monocyclic, bicyclic, and polycyclic aromatic hydrocarbons; azaarenes, epoxides, chlorinated hydrocarbons, and other aliphatic and aromatic compounds. The types of reactions performed by A. niger, although not unique to this species, are extremely diverse. They include hydroxylation, oxidation of various functional groups, reduction of double bonds, demethylation, sulfation, epoxide hydrolysis, dechlorination, ring cleavage, and conjugation. Some of the products may be useful as new investigational drugs or chemical intermediates.

Pleiotropic and epistatic behavior of a ring-hydroxylating oxygenase system in the polycyclic aromatic hydrocarbon metabolic network from Mycobacterium vanbaalenii PYR-1.

Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and its pleiotropic and epistatic behavior(s) responsible for low-molecular-weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparisons using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity directed toward three-ring PAHs. Structure analysis and PAH substrate docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taking the findings together, the Pdo system is an RHO system with the pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also crucial for the metabolic quality and quantity of the PAH-MN.

Identification of the enzyme responsible for N-acetylation of norfloxacin by Microbacterium sp. Strain 4N2-2.

Microbacterium sp. 4N2-2, isolated from a wastewater treatment plant, converts the antibacterial fluoroquinolone norfloxacin to N-acetylnorfloxacin and three other metabolites. Because N-acetylation results in loss of antibacterial activity, identification of the enzyme responsible is important for understanding fluoroquinolone resistance. The enzyme was identified as glutamine synthetase (GS); N-acetylnorfloxacin was produced only under conditions associated with GS expression. The GS gene (glnA) was cloned, and the protein (53 kDa) was heterologously expressed and isolated. Optimal conditions and biochemical properties (K(m) and V(max)) of purified GS were characterized; the purified enzyme was inhibited by Mn(2+), Mg(2+), ATP, and ADP. The contribution of GS to norfloxacin resistance was shown by using a norfloxacin-sensitive Escherichia coli strain carrying glnA derived from Microbacterium sp. 4N2-2. The GS of Microbacterium sp. 4N2-2 was shown to act as an N-acetyltransferase for norfloxacin, which produced low-level norfloxacin resistance. Structural and docking analysis identified potential binding sites for norfloxacin at the ADP binding site and for acetyl coenzyme A (acetyl-CoA) at a cleft in GS. The results suggest that environmental bacteria whose enzymes modify fluoroquinolones may be able to survive in the presence of low fluoroquinolone concentrations.

Photobleaching with phloxine B sensitizer to reduce food matrix interference for detection of Escherichia coli serotype O157:H7 in fresh spinach by flow cytometry.

A flow cytometric method (RAPID-B™) with detection sensitivity of one viable cell of Escherichia coli serotype O157:H7 in fresh spinach (Spinacia oleracea) was developed and evaluated. The major impediment to achieving this performance was mistaking autofluorescing spinach particles for tagged target cells. Following a 5 h non-selective enrichment, artificially inoculated samples were photobleached, using phloxine B as a photosensitizer. Samples were centrifuged at high speed to concentrate target cells, then gradient centrifuged to separate them from matrix debris. In external laboratory experiments, RAPID-B and the reference method both correctly detected E. coli O157:H7 at inoculations of ca. 15 cells. In a follow-up study, after 4 cell inoculations of positives and 6 h enrichment, RAPID-B correctly identified 92% of 25 samples. The RAPID-B method limit of detection (LOD) was one cell in 25 g. It proved superior to the reference method (which incorporated real time-PCR, selective enrichment, and culture plating elements) in accuracy and speed.

CPGminer: An Interactive Dashboard to Explore the Genomic Features and Taxonomy of Complete Prokaryotic Genomes.

Prokaryotes, the earliest forms of life on Earth, play crucial roles in global biogeochemical processes in virtually all ecosystems. The ever-increasing amount of prokaryotic genome sequencing data provides a wealth of information to examine fundamental and applied questions through systematic genome comparison. Genomic features, such as genome size and GC content, and taxonomy-centric genomic features of complete prokaryotic genomes (CPGs) are crucial for various fields of microbial research and education, yet they are often overlooked. Additionally, creating systematically curated datasets that align with research concerns is an essential yet challenging task for wet-lab researchers. In this study, we introduce CPGminer, a user-friendly tool that allows researchers to quickly and easily examine the genomic features and taxonomy of CPGs and curate genome datasets. We also provide several examples to demonstrate its practical utility in addressing descriptive questions.

Microbial transformation of azaarenes and potential uses in pharmaceutical synthesis.

Pyridine, quinoline, acridine, indole, carbazole, and other heterocyclic nitrogen-containing compounds (azaarenes) can be transformed by cultures of bacteria and fungi to produce a variety of new derivatives, many of which have biological activity. In many cases, the microbial biotransformation processes are regio- and stereoselective so that the transformation products may be useful for the synthesis of new candidate drugs.

Comparison of the effects of antimicrobial agents from three different classes on metabolism of isoflavonoids by colonic microflora using Etest strips.

Daidzein (4',7-dihydroxyisoflavone), a phytoestrogen found in soybeans mainly in the form of its glycoside daidzin, is metabolized by colonic bacteria to compounds with altered estrogenic activities, which may affect human health. Antibacterial agents used for the treatment of infections can alter the composition of bacterial populations in the colon and therefore can affect daidzein metabolism. To rapidly detect the effects of different concentrations of antibiotics on daidzein metabolism by colonic bacteria of monkeys and identify the subpopulation involved in daidzein metabolism, Etest strips containing antibacterial agents from three classes (tetracyclines, fluoroquinolones, and ß-lactams) were used to eliminate the colonic bacteria that were susceptible to 0-32 µg/ml of each antibacterial agent and test the surviving bacteria for their ability to metabolize daidzein. The metabolism of daidzein by the colonic microflora was measured before and after the colonic bacterial population was exposed to antibacterial agents. The metabolites were detected by high performance liquid chromatography and mass spectrometry after incubation of the cultures for various times. Exposure of colonic microflora to antibiotics had various effects on daidzein metabolism. Tetracycline completely removed the bacteria metabolizing daidzein, metabolism of daidzein was not changed in cultures of bacteria after ceftriaxone treatment, and ciprofloxacin enriched for the bacteria metabolizing daidzein. In liquid cultures treated with various concentrations of ciprofloxacin, 4 µg/ml of ciprofloxacin favored the growth of bacteria that metabolized daidzein. This is the first time in which the Etest has been used to show that, whereas some antibiotics eliminate phytoestrogen-metabolizing bacteria in colonic microflora, others enrich them by eliminating the non-metabolizing strains in the population.

Microbial transformations of antimicrobial quinolones and related drugs.

The quinolones are an important group of synthetic antimicrobial drugs used for treating bacterial diseases of humans and animals. Microorganisms transform antimicrobial quinolones (including fluoroquinolones) and the pharmacologically related naphthyridones, pyranoacridones, and cinnolones to a variety of metabolites. The biotransformation processes involve hydroxylation of methyl groups; hydroxylation of aliphatic and aromatic rings; oxidation of alcohols and amines; reduction of carboxyl groups; removal of methyl, carboxyl, fluoro, and cyano groups; addition of formyl, acetyl, nitrosyl, and cyclopentenone groups; and cleavage of aliphatic and aromatic rings. Most of these reactions greatly reduce or eliminate the antimicrobial activity of the quinolones.

Reduction of food matrix interference by a combination of sample preparation and multi-dimensional gating techniques to facilitate rapid, high sensitivity analysis for Escherichia coli serotype O157 by flow cytometry.

Escherichia coli serotype O157 strains, which may be found in foods, often produce enterohemorrhagic toxins. The research goal was to facilitate rapid, sensitive detection in foods of E. coli serotype O157 by flow cytometry. Sample preparation methods were developed for potential use in 15 foods. Combined with multi-dimensional gating, these methods decreased time-to-results (TTR) for determination of low-level contamination. They mitigated the effects of interfering food components, concentrated cells for analysis without growth or, when necessary, used short-term incubation. The results showed qualitative analysis that was equivalent to culture plating in accuracy and superior in sensitivity and speed. Preparation time was 10-30 min per sample and detection took 3-4 min. Culture growth, if required, took an additional 4-6 h. A protocol for raw spinach analysis, using 4 h culture incubation, was 94% correct with one false negative for a low level inoculation. Its projected limit-of-detection (LOD) was 1 viable cell per 25 g of spinach, based on an average of 28 counts detected after growth and an estimated counts-to-threshold (C/T) ratio of 1.3. The results suggested potential uses for regulatory screening and food industry process control.

Modification of norfloxacin by a Microbacterium sp. strain isolated from a wastewater treatment plant.

Antimicrobial residues found in municipal wastewater may increase selective pressure on microorganisms for development of resistance, but studies with mixed microbial cultures derived from wastewater have suggested that some bacteria are able to inactivate fluoroquinolones. Medium containing N-phenylpiperazine and inoculated with wastewater was used to enrich fluoroquinolone-modifying bacteria. One bacterial strain isolated from an enrichment culture was identified by 16S rRNA gene sequence analysis as a Microbacterium sp. similar to a plant growth-promoting bacterium, Microbacterium azadirachtae (99.70%), and a nematode pathogen, "M. nematophilum" (99.02%). During growth in medium with norfloxacin, this strain produced four metabolites, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) analyses as 8-hydroxynorfloxacin, 6-defluoro-6-hydroxynorfloxacin, desethylene norfloxacin, and N-acetylnorfloxacin. The production of the first three metabolites was enhanced by ascorbic acid and nitrate, but it was inhibited by phosphate, amino acids, mannitol, formate, and thiourea. In contrast, N-acetylnorfloxacin was most abundant in cultures supplemented with amino acids. This is the first report of defluorination and hydroxylation of a fluoroquinolone by an isolated bacterial strain. The results suggest that some bacteria may degrade fluoroquinolones in wastewater to metabolites with less antibacterial activity that could be subject to further degradation by other microorganisms.

Biotransformation of acridine by Mycobacterium vanbaalenii.

Cultures of Mycobacterium vanbaalenii strain PYR-1 in a liquid medium were exposed to the toxic environmental contaminant acridine (260 microM). After incubation for 7 d, the cultures were extracted with ethyl acetate. Metabolites were purified using high-performance liquid chromatography and analyzed by mass spectrometry and 1H nuclear magnetic resonance spectroscopy. Four metabolites, 9,10-dihydroacridine, 4-hydroxyacridine, acridine cis-1 ,2-dihydrodiol, and acridin-9(10H)-one, were identified.

Effects of nano-hydroxyapatite on the formation of biofilms by Streptococcus mutans in two different media.

OBJECTIVES: The aim of this study was to examine the effect of nano-hydroxyapatite (nHA) on biofilm formation by Streptococcus mutans, which is actively involved in the initiation of dental caries. DESIGN: The effects of nHA on growth and biofilm formation by S. mutans were investigated in two media: a saliva analog medium, basal medium mucin (BMM); and a nutrient-rich medium, brain heart infusion (BHI); in the presence and absence of sucrose. RESULTS: Sucrose enhanced the growth of S. mutans in both media. In the presence of sucrose, nHA enhanced bacterial growth and biofilm formation more in BMM medium than in BHI. nHA also affected the transcription of glucosyltransferase (gtf) genes and production of polysaccharide differently in the two media. In BHI medium, the transcription of all three gtf genes, coding for enzymes that synthesize soluble and insoluble glucans from sucrose, was increased more than 3-fold by nHA. However, in BMM medium, only the transcription of gtfB and gtfC, coding for insoluble glucans, was substantially enhanced by nHA. CONCLUSIONS: nHA appeared to enhance biofilm formation by increasing glucosyltransferase transcription, which resulted in an increase in production of insoluble glucans. This effect was influenced by the growth conditions.

Transformation of N-phenylpiperazine by mixed cultures from a municipal wastewater treatment plant.

Samples from a wastewater treatment plant were used as inocula for mixed cultures dosed with N-phenylpiperazine (NPP), a model compound containing the piperazine ring found in many fluoroquinolones. Chemical analyses showed that NPP (50 mg liter(-1)) disappeared in 12 days, with the appearance of a transient metabolite and two nitrosated compounds.

Biotransformation of flumequine by the fungus Cunninghamella elegans.

The metabolism of the antibacterial fluoroquinolone drug flumequine by Cunninghamella elegans was investigated using cultures grown in Sabouraud dextrose broth with 308microM flumequine. The cultures were extracted with ethyl acetate; metabolites were separated by high-performance liquid chromatography and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Flumequine was transformed to two diastereomers of 7-hydroxyflumequine (23 and 43% of the total chromatographic peak area at 280nm) and 7-oxoflumequine (11% of the total peak area). This is the first time that the two 7-hydroxy diastereomers have been characterized structurally; the hydroxyflumequines are known to have less antimicrobial activity than flumequine.

Rapid phenotypic characterization of Salmonella enterica strains by pyrolysis metastable atom bombardment mass spectrometry with multivariate statistical and artificial neural network pattern recognition.

Pyrolysis mass spectrometry was investigated for rapid characterization of bacteria. Spectra of Salmonella were compared to their serovars, pulsed-field gel electrophoresis (PFGE) patterns, antibiotic resistance profiles, and MIC values. Pyrolysis mass spectra generated via metastable atom bombardment were analyzed by multivariate principal component-discriminant analysis and artificial neural networks (ANNs). Spectral patterns developed by discriminant analysis and tested with Leave-One-Out (LOO) cross-validation distinguished Salmonella strains by serovar (97% correct) and by PFGE groups (49%). An ANN model of the same PFGE groups was cross-validated, using the LOO rule, with 92% agreement. Using an ANN, thirty previously unseen spectra were correctly classified by serotype (97%) and at the PFGE level (67%). Attempts by ANN to model spectra grouped by resistance profile-but ignoring PFGE or serotype-failed (10% correct), but ANNs differentiating ten samples of the same serotype/PFGE class were more successful. To assess the information content of PyMS data serendipitously associated with or directly related to resistance character, the ten isolates were grouped into four, three, or two categories. The four categories corresponded to four resistance profiles. The four class and three class ANNs showed much improved but insufficient modeling power. The two-class ANN and a corresponding multivariate model maximized inferential power for a coarse antibiotic-resistance-related distinction. They each cross-validated by LOO at 90%. This is the first direct correlation of pyrolysis metastable atom bombardment mass spectrometry with immunological (e.g. serology) or molecular biology (e.g. PFGE) based techniques.