To combat the spread of antibiotic resistance, methods that quantitatively assess the metabolism-inhibiting effects of drugs in a rapid and culture-independent manner are urgently needed. Here using four oral bacteria as models, we show that heavy water (DO)-based single-cell Raman microspectroscopy (DO-Raman) can probe bacterial response to different drugs using the Raman shift at the C-D (carbon-deuterium vibration) band in 2040 to 2300 cm as a universal biomarker for metabolic activity at single-bacterial-cell resolution. The "minimum inhibitory concentration based on metabolic activity" (MIC-MA), defined as the minimal dose under which the median ΔC-D-ratio at 8 h of drug exposure is ≤0 and the standard deviation (SD) of the ΔC-D ratio among individual cells is ≤0.005, was proposed to evaluate the metabolism-inhibiting efficacy of drugs. In addition, heterogeneity index of MIC-MA (MIC-MA-HI), defined as SD of C-D ratio among individual cells, quantitatively assesses the among-cell heterogeneity of metabolic activity after drug regimens. When exposed to 1× MIC of sodium fluoride (NaF), 1× MIC of chlorhexidine (CHX), or 60× MIC of ampicillin, the cariogenic oral pathogen Streptococcus mutans UA159 ceased propagation yet remained metabolically highly active. This underscores the advantage of MIC-MA over the growth-based MIC in being able to detect the "nongrowing but metabolically active" (NGMA) cells that underlie many latent or recurring infections. Moreover, antibiotic susceptible and resistant S. mutans strains can be readily discriminated at as early as 0.5 h. Thus, DO-Raman can serve as a universal method for rapid and quantitative assessment of antimicrobial effects based on general metabolic activity at single-cell resolution.

Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 M and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 ؎ 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 ؎ 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 ؎ 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 ؎ 1, 3.1 ؎ 0.3, and 0.26 ؎ 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 ؎ 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 ؎ 0.07 and 1.5 ؎ 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 ؎ 0.8, 4.0 ؎ 0.6, and 2.4 ؎ 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 M).

The interactions between microorganisms driven by substrate metabolism and energy flow are important to shape diversity, abundance, and structure of a microbial community. Single cell technologies are useful tools for dissecting the functions of individual members and their interactions in microbial communities. Here, we developed a novel Raman stable isotope probing (Raman-SIP), which uses Raman microspectroscopy coupled with reverse and DO colabeling to study metabolic interactions in a two-species community consisting of Acinetobacter baylyi ADP1 and Escherichia coli DH5α-GFP. This Raman-SIP approach is able to detect carbon assimilation and general metabolic activity simultaneously. Taking advantage of Raman shift of single cell Raman spectra (SCRS) mediated by incorporation of stable-isotopic substrates, Raman-SIP with reverse labeling has been applied to detect initially C-labeled bands of ADP1 SCRS reverting back toC positions in the presence of C citrate. Raman-SIP with DO labeling has been employed to probe metabolic activity of single cells without the need of cell replication. Our results show that E. coli alone in minimal medium with citrate as the sole carbon source had no metabolic activity, but became metabolically active in the presence of ADP1. Mass spectrometry-based metabolite footprint analysis suggests that putrescine and phenylalanine excreted by ADP1 cells may support the metabolic activity of E. coli. This study demonstrates that Raman-SIP with reverse labeling would be a useful tool to probe metabolism of any carbon substrate, overcoming limitations when stable isotopic substrates are not readily available. It is also found that Raman-SIP with DO labeling is a sensitive and reliable approach to distinguish metabolically active cells but not quiescent cells. This novel approach extends the application of Raman-SIP and demonstrates its potential application as a valuable strategic approach for probing cellular metabolism, metabolic activity, and interactions in microbial communities at the single cell level.

Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent V max values of 6.6 ؎ 0.9 to 10.7 ؎ 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ؎ 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase ( . Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/ A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.

After discovering that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes nitrobenzene to 4-nitrocatechol, albeit at a very low rate, this reaction was improved using directed evolution and saturation mutagenesis. Screening 550 colonies from a random mutagenesis library generated by error-prone PCR of tmoAB using Escherichia coli TG1/pBS(Kan)T4MO on agar plates containing nitrobenzene led to the discovery of nitrocatechol-producing mutants. One mutant, NB1, contained six amino acid substitutions (TmoA Y22N, I84Y, S95T, I100S, S400C; TmoB D79N). It was believed that position I100 of the alpha subunit of the hydroxylase (TmoA) is the most significant for the change in substrate reactivity due to previous results in our lab with a similar enzyme, toluene ortho-monooxygenase of Burkholderia cepacia G4. Saturation mutagenesis at this position resulted in the generation of two more nitrocatechol mutants, I100A and I100S; the rate of 4-nitrocatechol formation by I100A was more than 16 times higher than that of wild-type T4MO at 200 microM nitrobenzene (0.13 +/- 0.01 vs. 0.008 +/- 0.001 nmol/min.mg protein). HPLC and mass spectrometry analysis revealed that variants NB1, I100A, and I100S produce 4-nitrocatechol via m-nitrophenol, while the wild-type produces primarily p-nitrophenol and negligible amounts of nitrocatechol. Relative to wild-type T4MO, whole cells expressing variant I100A convert nitrobenzene into m-nitrophenol with a Vmax of 0.61 +/- 0.037 vs. 0.16 +/- 0.071 nmol/min.mg protein and convert m-nitrophenol into nitrocatechol with a Vmax of 3.93 +/- 0.26 vs. 0.58 +/- 0.033 nmol/min.mg protein. Hence, the regiospecificity of nitrobenzene oxidation was changed by the random mutagenesis, and this led to a significant increase in 4-nitrocatechol production. The regiospecificity of toluene oxidation was also altered, and all of the mutants produced 20% m-cresol and 80% p-cresol, while the wild-type produces 96% p-cresol. Interestingly, the rate of toluene oxidation (the natural substrate of the enzyme) by I100A was also higher by 65% (7.2 +/- 1.2 vs. 4.4 +/- 0.3 nmol/min mg protein). Homology-based modeling of TmoA suggests reducing the size of the side chain of I100 leads to an increase in the width of the active site channel, which facilitates access of substrates and promotes more flexible orientations.

A two-phase system is developed here for converting: (1) benzene to phenol and (2) naphthalene to 2-naphthol, using whole cells expressing wild-type toluene 4-monooxygenase (T4MO) and the alpha subunit variant TmoA I100A from Pseudomonas mendocina KR1. Using the T4MO TmoA I100A variant, the solubility of naphthalene was enhanced and the toxicity of the naphthols was prevented by the use of a water/dioctyl phthalate (80:20, vol%) system which yielded 21-fold more 2-naphthol. More than 99% 2-naphthol was extracted to the dioctyl phthalate phase, dihydroxynaphthalene formation was prevented, 92% 2-naphthol was formed, and 12% naphthalene was converted. Similarly, using 50 vol% dioctyl phthalate, an initial concentration of 3.0 g l(-1) (39 mM), and wild-type T4MO, a 51+/-9% conversion of benzene was obtained and phenol was produced at a purity of 97%. Relative to the one-phase system, there was a 12-fold reduction in the formation of the byproduct catechol.

A primary goal of protein engineering is to control catalytic activity. Here we show that through mutagenesis of three active site residues, the catalytic activity of a multicomponent monooxygenase is altered so that it hydroxylates all three positions of toluene as well as both positions of naphthalene. Hence, for the first time, an enzyme has been engineered so that its regiospecific oxidation of a substrate can be controlled. Through the A107G mutation in the ␣-subunit of toluene para-monooxygenase, a variant was formed that hydroxylated toluene primarily at the ortho-position while converting naphthalene to 1-naphthol. Conversely, the A107T variant produced >98% p-cresol and p-nitrophenol from toluene and nitrobenzene, respectively, as well as produced 2-naphthol from naphthalene. The mutation I100S/G103S produced a toluene para-monooxygenase variant that formed 75% m-cresol from toluene and 100% m-nitrophenol from nitrobenzene; thus, for the first time a true meta-hydroxylating toluene monooxygenase was created.Enzymes are attractive catalysts for chemical synthesis because of their exceptional enantio-and regioselectivities; hence, enzymes can be used in both simple and complex syntheses without the need for the blocking and deblocking steps often found in their organic counterparts (1, 2). The number of biocatalytic processes that are being performed on a large scale is rapidly increasing, and it is projected that this growth will continue (3, 4) to become 30% of the chemical business by the year 2050 (5). Although the majority of currently used industrial enzymes are hydrolases (4), there is growing interest in the application of oxygenases (5). Oxidative biotransformations use oxygen as an inexpensive, environmentally friendly oxidant in contrast to toxic chemical oxidants, and they exceed their chemical equivalent in regiospecificity and enantioselectivity (6, 7). Among bacterial oxygenases, toluene monooxygenases have a lot of potential for bioremediation and chemical synthesis, as we have used mutagenesis to make, among other things, variants that remediate one of the world's worst pollutants (trichloroethylene) (8), form a rainbow of colors from indole with a single enzyme, and make the anti-cancer compound indirubin (9). These variants also make useful compounds like 1-naphthol (15,000 ton/year market) (8), nitrohydroquinone (precursor for therapeutics for Alzheimer and Parkinson disease) (10), and 4-nitrocatechol (inhibitor of nitric-oxide synthase) (11). For these industrial applications, control of regiospecific oxidation of aromatics is of vital importance. Further, regiospecific oxidation of toluene, substituted benzenes, and naphthalene by these monooxygenases presents an important challenge both in terms of the three distinct regiospecific oxidations possible for the initial hydroxylation of toluene as well as in terms of the usefulness of both the mono-and dihydroxylated products.Toluene para-monooxygenase (TpMO, 1 formerly known as T3MO) of Ralstonia pickettii PKO1 is a soluble, non-heme, ...

The regiospecific oxidation of the polycyclic aromatic hydrocarbons naphthalene and fluorene was examined with Escherichia coli strains expressing wildtype toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1, toluene para-monooxygenase (TpMO) from Ralstonia pickettii PKO1, toluene ortho-monooxygenase (TOM) from Burkholderia cepacia G4, and toluene/ortho-xylene monooxygenase (ToMO) from P. stutzeri OX1. T4MO oxidized toluene (12.1+/-0.8 nmol/min/mg protein at 109 microM), naphthalene (7.7+/-1.5 nmol/min/mg protein at 5 mM), and fluorene (0.68+/-0.04 nmol/min/mg protein at 0.2 mM) faster than the other wildtype enzymes (2-22-fold) and produced a mixture of 1-naphthol (52%) and 2-naphthol (48%) from naphthalene, which was successively transformed to a mixture of 2,3-, 2,7-, 1,7-, and 2,6-dihydroxynaphthalenes (7%, 10%, 20%, and 63%, respectively). TOM and ToMO made 1,7-dihydroxynaphthalene from 1-naphthol, and ToMO made a mixture of 2,3-, 2,6-, 2,7-, and 1,7-dihydroxynaphthalene (26%, 22%, 1%, and 44%, respectively) from 2-naphthol. TOM had no activity on 2-naphthol, and T4MO had no activity on 1-naphthol. To take advantage of the high activity of wildtype T4MO but to increase its regiospecificity on naphthalene, seven engineered enzymes containing mutations in T4MO alpha hydroxylase TmoA were examined; the selectivity for 2-naphthol by T4MO I100A, I100S, and I100G was enhanced to 88-95%, and the selectivity for 1-naphthol was enhanced to 87% and 99% by T4MO I100L and G103S/A107G, respectively, while high oxidation rates were maintained except for G103S/A107G. Therefore, the regiospecificity for naphthalene oxidation was altered to practically pure 1-naphthol or 2-naphthol. All four wildtype monooxygenases were able to oxidize fluorene to different monohydroxylated products; T4MO oxidized fluorene successively to 3-hydroxyfluorene and 3,6-dihydroxyfluorene, which was confirmed by gas chromatography-mass spectrometry and 1H nuclear magnetic resonance analysis. TOM and its variant TomA3 V106A oxidize fluorene to a mixture of 1-, 2-, 3-, and 4-hydroxyfluorene. This is the first report of using enzymes to synthesize 1-, 3-, and 4-hydroxyfluorene, and 3,6-dihydroxyfluorene from fluorene as well as 2-naphthol and 2,6-dihydroxynaphthalene from naphthalene.