INTRODUCTION

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Oligonucleotide hybridization probes targeting the small subunit rRNA (16S and 16S-like rRNAs) and the larger rRNA of the large ribosomal subunit (23S and 23S-like rRNAs) have made it possible to determine the composition of microbial communities and to estimate the activity of microbial populations in numerous environments, including activated sludge wastewater treatment systems (for recent reviews, see references 5 and 26). For example, fluorescence in situ hybridization (FISH) has been used to quantify the biomass of specific filamentous microorganisms in activated sludge using a relationship between the number and length of individual target cells and biomass concentration (10). In addition, membrane hybridizations have been used to measure the activity of microbial populations in foaming activated sludge (11) and in activated sludge systems operated for enhanced biological phosphorus removal (20). Although these approaches may not work for all microbial populationssuch as metabolically active microorganisms with low rRNA content (19) and metabolically inactive microorganisms with a high residual rRNA content (25, 32)oligonucleotide hybridization probes targeting rRNA have been used successfully to study the microbial ecology of activated sludge (5).

To take full advantage of engineering efforts in rational design and optimization of activated sludge systems, results from oligonucleotide probe hybridizations remain to be interfaced with representations of microbial biomass and activity used in mathematical models of microbial growth and substrate utilization (21). This will only become possible when rapid and reliable methods for quantifying changes in the activity of microbial populations become available. One approach, initially suggested by DeLong and coworkers (12), is based upon determining the relationship between cellular ribosome content and growth rate of a target population. When this information is available, FISH signal intensity can be quantified with digital microscopy to determine the in situ cellular growth rate in environmental samples. This approach has been used to estimate the in situ cellular growth rates of a sulfate-reducing bacterium in biofilms (23), of Pseudomonas fluorescens in environmental mesocosms (6), of Pseudomonas strain B13(FR1) in marine microcosms (16), and of Pseudomonas putida in biofilms (18). Results of FISH targeting rRNA have been combined with a model of microbial growth and substrate utilization to study the zero-order degradation of toluene by P. putida (18, 22).

Recently, Cangelosi and Brabant (8) suggested an alternative approach for measuring in situ growth activity. Instead of using oligonucleotide hybridization probes to quantify the levels of rRNA, they designed oligonucleotide probes to monitor the expression of rRNA genes. In most bacteria, the 16S, 23S, and 5S rRNAs and a variety of tRNA genes are transcribed from polycistronic rrn operons (for a review, see reference 29). RNase III subsequently cleaves the full-length transcript into precursor RNA fragments. Each precursor RNA fragment contains a region of nucleotides at the 5' end followed by a single mature RNA and an additional region of nucleotides at the 3' end (15). For example, whereas the mature 16S rRNA has 1,542 nucleotides, the precursor 16S rRNA from rrnB in Escherichia coli contains 1,731 nucleotides, with 146 nucleotides at the 5' terminus of the mature 16S rRNA and 43 nucleotides at the 3' terminus (7, 33).

Cangelosi and Brabant (8) used oligonucleotide probes targeting the 5' and 3' regions of the precursor 16S rRNA of E. coli to show that the levels of precursor 16S rRNA vary significantly over growth phases. Their results indicate that, at least for some microbial populations, quantification of the expression of rrn genes can be used in place of direct measurement of cellular rRNA to monitor microbial growth in situ. By combining oligonucleotide probes targeting 16S rRNA and precursor 16S rRNA, the microbial community composition and growth activities of specific microbial populations may be measured in the same sample. In this study, we report on the design and characterization of oligonucleotide probes targeting precursor 16S rRNA sequences of Acinetobacter spp. as a model system for evaluating this approach in activated sludge. Subsequently, we used these probes to monitor changes in precursor 16S rRNA and total 16S rRNA levels during batch growth of a pure culture of Acinetobacter calcoaceticus and to monitor growth activity of Acinetobacter spp. within lab- and full-scale activated sludge wastewater treatment systems.

	MATERIALS AND METHODS

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Batch growth of microorganisms and sampling. A. calcoaceticus ATCC 23055^T and E. coli ATCC 11775^T were cultured aerobically in Luria-Bertani (LB) medium (27) at 35°C on rotary shakers. Overnight cultures were diluted 20-fold into fresh LB medium or wastewater filtered through a 0.2-µm-pore-size filter (Nalgene, Rochester, N.Y.). Each batch growth study was conducted in duplicate. At sampling time points, the optical density of the culture was determined spectrophotometrically at a wavelength of 600 nm (OD₆₀₀). Two samples (2 ml each) from each batch culture were removed and centrifuged at 14,000 × g, and the supernatant was decanted. One sample was immediately frozen in an ethanol-dry ice bath, and RNA was extracted by a bead-beating, low-pH, phenol-chloroform protocol (26, 30). The second sample was fixed in 4% paraformaldehyde for 1 h at room temperature and subsequently stored in 50% ethanol in phosphate-buffered saline (130 mM NaCl, 10 mM sodium phosphate buffer [pH 7.2]) at 20°C (9). To prepare pure cultures with a high fraction of precursor rRNA, overnight cultures of E. coli and A. calcoaceticus, diluted 20-fold into fresh LB medium, were treated with chloramphenicol (final concentration of 20 mg/liter) (Sigma, St. Louis, Mo.) after 1 h of growth at 35°C (17). The cultures remained at 35°C for 1 h, and samples were taken for extraction of RNA and fixation for FISH.

Probe synthesis and labeling. For membrane hybridizations, oligonucleotide probes, synthesized by the University of Illinois Biotechnology Facility, were 5' end labeled with [-³²P]ATP (24). Oligonucleotides for FISH were conjugated with the cyanine dye Cy3 or Cy5 before purification with high-performance liquid chromatography (Interactive, Germany). Fluorescently labeled probes were diluted to 50 mg/liter with H₂O and stored in 50-µl aliquots at 20°C in the dark. Probe and target sequences are listed in Table 1.

Preparation of recombinant rDNA plasmids. Genomic DNA was extracted from overnight cultures of E. coli and A. calcoaceticus by a microwave lysis procedure (11). PCR amplification of near-complete 16S ribosomal DNA (rDNA) and the intergenic spacer region was conducted with primer pair S-D-Bact-0011-a-S-17 (5'-GTTTGATCCTGGCTCAG-3') and L-Sc-gProt-1207-a-A-17 (5'-GCCTTCCCACATCGTTT-3') using standard methods (11). The PCR products were subcloned with a TA Cloning Kit (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. Recombinant plasmids were recovered with a lysozyme and sodium dodecyl sulfate (SDS) lysis and a phenol-chloroform extraction (27). Endonuclease restriction digests using EcoRI confirmed correct recombinants, and the recombinant rDNA plasmids were stored at 20°C in Tris-EDTA buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA [pH 8.0]).

Determination of optimal wash temperature. For temperature of dissociation (T_d) studies, total RNA extracted from chloramphenicol-treated cells and recombinant rDNA plasmid were denatured in 3 volumes of 2% glutaraldehyde, diluted to 0.5 mg/liter with dilution water (24) without poly(A), and 50 ng of each nucleic acid sample was applied by slot blotting to Magna Charge membranes (Micron Separation, Inc., Westboro, Mass.) (24). The membranes were baked for 2 h at 80°C, prehybridized for 2 h at 40°C, hybridized for 12 h at 30°C, and initially washed twice with 100 ml of wash buffer (1% SDS and 1× SSC [0.15 M NaCl plus 0.015 M sodium citrate]) for 1 h each at 30°C (34). The T_d for each probe was experimentally determined by an elution method as previously described (34).

Quantitative membrane hybridizations. For membrane hybridizations, RNA extracts were denatured as described above, 200 ng of RNA was applied in triplicate to Magna Charge membranes, and the membranes were baked for 2 h at 80°C (24). Prehybridizations were performed at 40°C for 2 to 6 h, and hybridizations took place at 30°C for 12 to 14 h. Membranes were initially washed twice for 1 h each in 100 ml of wash buffer at 30°C. Stringent washing was conducted for 30 min in 500 ml of fresh wash buffer at the experimentally determined wash temperature. The hybridization signal was quantified with an Electronic Autoradiography Instant Imager (Packard Instrument Company, Meriden, Conn.).

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Determination of optimal formamide stringency and FISH. Chloramphenicol-treated cultures, fixed in 4% paraformaldehyde, were applied in a sample well on a Heavy-Teflon-Coated microscope slide (Cel-Line Associates, New Field, N.J.) and air dried. After dehydration with an increasing ethanol series (50, 80, and 95% [vol/vol] ethanol, 1 min each), each sample well was covered with 9 µl of hybridization buffer (0% [vol/vol] to 70% [vol/vol] formamide, 0.9 M NaCl, 100 mM Tris-HCl [pH 7.2], 0.1% SDS). A 1-µl portion of fluorescently labeled oligonucleotide probe (50 ng) was added to each well of the microscope slide. Hybridizations were conducted in a moisture chamber (4) for 1 to 2 h, in the dark, at 46°C. The slides were washed for 15 min at 48°C with 50 ml of prewarmed wash solution (500 to 20 mM NaCl, 20 mM Tris-HCl [pH 7.2], 0.1% SDS, 5 mM EDTA). Samples were counterstained with ice-cold, fresh 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining solution (1 mg of DAPI/liter) for 2 min, rinsed with ice-cold water, and rapidly air dried. Fixed, hybridized cells were mounted with a 1:4 (vol/vol) mixture of VectaShield (Vector Laboratories, Burlingame, Calif.) and Citifluor (Citifluor, Ltd., London, United Kingdom) immersion solutions and a coverslip. The optimal formamide stringency was determined with digital microscopy as previously described (9) with the modifications outlined below. Subsequent FISH experiments were performed using the optimal stringency.

Lab-scale activated sludge wastewater treatment systems. Three lab-scale activated sludge wastewater treatment systems, one completely mixed activated sludge (CMAS) system, and two sequencing batch reactors (SBR-1 and SBR-2) were operated with a hydraulic retention time (HRT) of 10 h and a mean cell residence time (MCRT) of 5 days. The CMAS system consisted of an aeration basin of 10 liters and a clarifier of 3 liters. This system was operated with an influent flow rate of 1.3 liter/h and a recycle ratio of 0.25. The mixed liquor volatile suspended solids (MLVSS) in the CMAS system was 987 mg/liter, and the food-to-microorganism ratio (F/M) was 0.11 mg of soluble chemical oxygen demand (S-COD) mg of volatile suspended solids (VSS)¹ day¹. SBR-1 was operated with an 8-h cycle time consisting of 20 min of filling with aeration, 340 min of aeration, 100 min of settling, and 20 min of decanting (4 liters of effluent were decanted). SBR-2 was operated with a 4-h cycle time consisting of 10 min of filling with aeration, 170 min of aeration, 50 min of settling, and 10 min of decanting (2 liters of effluent were decanted). The MLVSS in SRB-1 and SBR-2 were 673 mg/liter, and the F/M was 0.162 mg of S-COD mg of VSS¹ day¹. The systems were seeded with activated sludge from the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant (UCSD, NEWWTP), and were used to treat primary clarifier effluent collected from the UCSD, NEWWTP. After 20 days of operation, the systems were sampled over an 8-h period. The MLVSS levels were determined according to standard methods (12), and the S-COD levels of the influent and mixed liquor were determined with Hach Tests (Hach, Loveland, Colo.) according to the manufacturer's instructions.

Full-scale activated sludge wastewater treatment system. The UCSD, NEWWTP, treats on average 5.7 × 10⁴ m³/day (15 million gallons/day) of municipal wastewater. The treatment plant reduces the average influent 5-day biochemical oxygen demand (BOD₅) of 150 mg of BOD₅/liter by 30% with primary clarification and by an additional 65% in a contact stabilization activated sludge system (T. Bachman, personal communication). The plant was operated with a target MCRT of 3.5 days and an F/M of approximately 0.2 g of BOD₅ g of SS¹ day¹. The average mixed liquor suspended solids (MLSS) concentration in the contact basin was 1.1 g/liter. The typical return activated sludge (RAS) SS concentration was 5 g/liter, which resulted in an MLSS concentration of approximately 4.5 g/liter in the re-aeration basin. Figure 1 shows a schematic layout of the UCSD, NEWWTP. Samples were collected from across the contact basin (sites B1, C6, and D1, Fig. 1), from the re-aeration basin (site A6, Fig. 1), and from the RAS line (just before site A6, Fig. 1) at 9:00 h and 13:00 h on 24 September 1998 and at 8:00 h and 12:00 h on 10 October 1998. 

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Schematic of the UCSD, NEWWTP. Samples from the contact basin were removed from sites B1, C6, and D1. The re-aeration basin was sampled at site A6, while the RAS line was sampled immediately before the re-aeration basin.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Probe specificity and hybridization stringency. Probes S-S-E.coli-1543-a-A-24 and S-S-E.coli-1543-b-A-24 were designed to target within the 3' region of the precursor 16S rRNA of operons rrnA, -C, -D, -E, -F, -G, and -H and rrnB of E. coli, respectively. Probe S-G-Acin-1543-a-A-24 was designed to target within the 3' region of the precursor 16S rRNA of the Acinetobacter genus. An advanced BLAST 2.0 (2) comparison of the oligonucleotide probe sequences, shown in Table 1, with the GenEMBL nonredundant nucleotide database was used to check the specificity of the probes. The BLAST analysis predicted that the oligonucleotide probes would hybridize with all intended targets. In addition, the analysis showed that the probes had at least two mismatches with nontarget sequences (with the exception of interoperon sequence similarity observed in E. coli).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Td studies for membrane hybridizations (a) and formamide stringency studies for FISH (b). The Td curves for probes S-S-E.coli-1543-a-A-24 (squares) and S-G-Acin-1543-a-A-24 (circles) were obtained using RNA extracted from chloramphenicol-treated E. coli and A. calcoaceticus (filled symbols) and recombinant rDNA plasmids (open symbols). Formamide stringency for probes S-S-E.coli-1543-a-A-24 (), S-S-E.coli-1543-b-A-24 (), and S-G-Acin-1543-a-A-24 () was obtained using chloramphenicol-treated E. coli and A. calcoaceticus fixed in 4% paraformaldehyde.

Batch growth of E. coli in LB medium. Figure 3 shows the changes in precursor 16S rRNA and total 16S rRNA during batch growth of E. coli in LB medium determined using membrane hybridizations. E. coli was diluted 20-fold into fresh LB medium from an overnight culture. The OD₆₀₀ of the overnight culture was 2.19 (Fig. 3). After diluting the culture, the OD₆₀₀ was determined to be 0.17. During the first 120 min of exponential growth, the OD₆₀₀ increased from 0.17 to 1.24. As the cultures entered stationary phase, the OD₆₀₀ increased from 1.24 to 2.59 after 480 min.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Results from membrane hybridizations of samples from batch growth of E. coli in LB medium. Probe S-S-E.coli-1543-a-A-24 was used to measure precursor 16S rRNA (), and probe S-*-Univ-1390-a-A-18 was used to measure total 16S rRNA (). The ratio of precursor 16S rRNA to total 16S rRNA () was determined using a reference dilution series of RNA extracted from chloramphenicol-treated E. coli. Biomass was estimated by measuring the OD600 (×).

Batch growth of A. calcoaceticus in LB medium. The results obtained with membrane hybridizations and FISH of batch cultures of A. calcoaceticus grown in LB medium are presented in Fig. 4a and 4b, respectively. The OD₆₀₀ of the overnight culture, 1.64, was lowered to an OD₆₀₀ of 0.07 after a 20-fold dilution into fresh medium (Fig. 4a). Throughout the study, the OD₆₀₀ increased and reached a value of 1.22 after 540 min of growth.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Results from membrane hybridizations (a) and FISH (b) of samples from batch growth of A. calcoaceticus in LB medium. Probe S-G-Acin-1543-a-A-24 was used to measure precursor 16S rRNA (). For membrane hybridizations, probe S-*-Univ-1390-a-A-18 was used to measure total 16S rRNA (), and for FISH, probe S-D-Bact-0338-a-A-18 was used to measure total 16S rRNA (). The ratio of precursor 16S rRNA to total 16S rRNA () was determined using a reference dilution series of RNA extracted from chloramphenicol-treated A. calcoaceticus. Biomass was estimated by measuring the OD600 (×).

Batch growth of A. calcoaceticus in filtered wastewater. An overnight culture of A. calcoaceticus, grown in LB medium, was diluted 20-fold with filtered (0.2-µm-pore-size filter) wastewater collected from the primary clarifier of the UCSD, NEWWTP. Results from measurements of OD₆₀₀ and from membrane hybridizations with probes targeting precursor 16S rRNA and total 16S rRNA are presented in Fig. 5. The OD₆₀₀ was 0.10 after the 20-fold dilution of the overnight culture into the filtered wastewater. During the first 45 min of growth, the OD₆₀₀ decreased slightly to a minimum of 0.02 after 45 min. Subsequently, the OD₆₀₀ increased to 0.17 540 min after the transfer, indicating an increase in the total biomass.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Results from membrane hybridizations of samples from batch growth of A. calcoaceticus in filtered wastewater. Probe S-G-Acin-1543-a-A-24 was used to measure precursor 16S rRNA (), and probe S-*-Univ-1390-a-A-18 was used to measure total 16S rRNA (). The ratio of precursor 16S rRNA to total 16S rRNA () was determined using a reference dilution series of RNA extracted from chloramphenicol-treated A. calcoaceticus. Biomass was estimated by measuring the OD600 (×).

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Results from FISH of samples from the batch growth of A. calcoaceticus in filtered wastewater. The fraction of total hybridized objects with an average gray value between the value reported and the next lowest value are plotted in histogram format against mean object gray values. The fractions of objects hybridized with the precursor 16S rRNA probe, S-G-Acin-1543-a-A-24, are shown in panels a to e. Results with probe S-D-Bact-0338-a-A-18, which targets total 16S rRNA, are shown in panels f to j. Samples removed from the overnight culture are shown in panels a and f. Panels b and g, c and h, d and i, and e and j show samples removed after 15, 180, 420, and 540 min of growth, respectively.

Simultaneous FISH with oligonucleotide probes targeting precursor 16S rRNA and total 16S rRNA. In Fig. 7, representative digital micrographs of samples of A. calcoaceticus transferred after overnight growth in LB medium to LB medium or to filtered wastewater are shown for various time points after transfer. The samples were simultaneously hybridized with probe S-G-Acin-1543-a-A-24, labeled with Cy5, and probe S-D-Bact-0338-a-A-18, labeled with Cy3. All digital micrographs were acquired using laser scanning confocal microscopy with constant settings (i.e., constant pinhole diameter, laser intensity, and electronic gain). Therefore, the hybridization signal from the precursor 16S rRNA probe, shown in red, and the hybridization signal from the total 16S rRNA probe, shown in green, can be compared for the images from the different samples. Figures 7a to d show images of A. calcoaceticus cultured in LB medium, while Fig. 7e to h are representative images of A. calcoaceticus cultured in filtered wastewater.

View larger version (22K): [in this window] [in a new window]   FIG. 7.   Representative digital micrographs from FISH with dual probes targeting precursor 16S rRNA (shown in red) and total 16S rRNA (shown in green). Results with samples from the batch growth of A. calcoaceticus in LB medium are shown in panels a to d. Panels e to h show results with samples from filtered wastewater. Samples removed from the overnight culture are shown in panels a and e. Panels b and f, c and g, and d and h show samples removed after 15, 180, and 540 min of growth, respectively. The bar is equal to 5 µm.

Precursor 16S rRNA and total 16S rRNA levels of Acinetobacter spp. in activated sludge wastewater treatment systems. Figure 8a shows the results of S-COD analyses for the three lab-scale activated sludge systems. The S-COD concentrations in SBR-1 increased to the S-COD concentration of the influent when 4 liters of influent was pumped into the reactor at the start of the operating cycle and rapidly decreased within the first 45 min of operation. The S-COD concentrations in SBR-2 showed a similar pattern, with an increase in S-COD levels after the addition of 2 liters of influent, and a rapid consumption of S-COD within the first 45 min of aeration. As expected, the S-COD concentrations in the CMAS system were relatively constant.

View larger version (32K): [in this window] [in a new window]   FIG. 8.   S-COD concentrations in the influent wastewater and samples of mixed liquor from the lab-scale activated sludge wastewater treatment systems and results from membrane hybridizations with samples from the mixed liquor using oligonucleotide probes targeting precursor 16S rRNA and total 16S rRNA. The results of S-COD concentrations are shown in panel a: CMAS (), SBR-1 (), and SBR-2 (×). The results of membrane hybridizations with samples from the CMAS are shown in panel b, while the results with samples from SBR-1 and SBR-2 are shown in panels c and d, respectively. Precursor 16S rRNA was hybridized with probe S-G-Acin-1543-a-A-24 (), while total 16S rRNA from Acinetobacter spp. and that from the total microbial community were measured with probes S-G-Acin-0659-a-A-24 () and S-*-Univ-1390-a-A-18 (), respectively. The ratio of precursor 16S rRNA to total 16S rRNA () was determined using a reference dilution series of RNA extracted from chloramphenicol-treated A. calcoaceticus.

View larger version (27K): [in this window] [in a new window]   FIG. 9.   Membrane hybridizations with samples from the UCSD, NEWWTP. Precursor 16S rRNA was measured using probe S-G-Acin-1543-a-A-24 (), while total 16S rRNA from Acinetobacter spp. and from the total microbial community was measured with probes S-G-Acin-0659-a-A-24 () and S-*-Univ-1390-a-A-18 (), respectively. The ratio of precursor 16S rRNA to total 16S rRNA () was determined using a reference dilution series of RNA extracted from chloramphenicol-treated A. calcoaceticus.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Probe design and characterization. A number of difficulties were encountered when designing precursor 16S rRNA probes. Searches of the GenEMBL database revealed few entries containing sufficient sequence information for the design of precursor 16S rRNA probes. Fortunately, four entries contained the sequences of the 3' region of the precursor 16S rRNA of Acinetobacter spp. (Table 1). As previously discussed, each precursor 16S rRNA is excised from the full-length rrn transcript by RNase III (15). The RNase III cleavage site is a sequence-independent, secondary-structure-dependent stem produced by intrastrand hybridization between the 5' and 3' regions of each precursor 16S rRNA molecule. Since no information was available regarding the location of the RNase III cleavage site for precursor 16S rRNA in Acinetobacter spp., probe S-G-Acin-1543-a-A-24 was designed based upon the location of previously reported precursor 16S rRNA probes targeting E. coli (8). In addition, although one species of Acinetobacter is known to have seven copies of the rrn operon (13), no information was available regarding interoperon sequence divergence for Acinetobacter spp. Therefore, the specificity of S-G-Acin-1543-a-A-24 could not be fully examined in this study.

Precursor rRNA levels in E. coli: comparison with previous results. Membrane hybridizations, using stringent experimental conditions, were used to monitor the levels of precursor 16S rRNA and total 16S rRNA during batch growth of E. coli in LB medium (Fig. 3). The rapid increase in the OD₆₀₀ values suggested that E. coli underwent a rapid transition from stationary to exponential growth phase. Thus, the extensive lag phase observed in similar experiments by Cangelosi and Brabant (8) was not found with our experimental conditions. In agreement with previous reports (8, 17), we observed an immediate increase in the levels of precursor 16S rRNA after transferring E. coli to fresh medium (Fig. 3). However, Cangelosi and Brabant (8) reported a 50-fold increase in the levels of precursor 16S rRNA, while we observed only a 12-fold increase (Fig. 3). A number of hypotheses are offered for this apparent discrepancy. First, results from our hybridizations should reflect the stringent experimental conditions used in our assays. The hybridization signals reported by Cangelosi and Brabant (8) with oligonucleotide probes targeting six of the seven rrn operons in E. coli (rrnA, -C, -D, -E, -F, -G, and -H), rrnB alone, and all seven rrn operons were similar for all three probes, suggesting that hybridization conditions were not stringent and that the observed signals corresponded to the levels of all seven transcripts. Thus, the 50-fold increase reported by Cangelosi and Brabant probably reflects the increased transcription of all seven rrn operons. In contrast, our use of stringent hybridization conditions with probe S-S-E.coli-1543-a-A-24 should limit our hybridization results to a signal corresponding to the level of transcripts corresponding to only six of the seven rrn operons (rrnA, -C, -D, -E, -F, -G, and -H). Second, Cangelosi and Brabant reported that the level of precursor 16S rRNA for their overnight culture was below the detection limit of their slot blot hybridization assay (8). Thus, the 50-fold increase they observed was calculated as the increase from the detection limit of their assay to the maximum hybridization signal observed. In our experiment, the hybridization signal of the precursor 16S rRNA for the overnight culture of E. coli was above the detection limit of our slot blot assay. Therefore, it is likely that the overnight cultures were prepared differently and that those differences are responsible for the differences between the results of Cangelosi and Brabant and our results. If the initial level of precursor 16S rRNA was significantly higher in our overnight culture than in the overnight culture used by Cangelosi and Brabant, the difference in hybridization results as well as the difference between the lengths of the observed lag phases may be explained.

Precursor rRNA levels in Acinetobacter spp. Acinetobacter spp. have been historically important in the study of activated sludge, especially in the area of enhanced biological phosphorus removal (20). Therefore, this genus was selected for further study. In agreement with the results for E. coli, precursor 16S rRNA levels increased rapidly at the onset of exponential growth and then declined as A. calcoaceticus entered the late-exponential-growth phase when cultured in LB medium (Fig. 4). The levels of total 16S rRNA reached a maximum during mid- to late-exponential-growth phase and declined throughout entry into stationary phase. These results suggest that the precursor 16S rRNA pool is much more sensitive to changes in growth phase than the total rRNA pool. Using membrane hybridizations, it was determined that the precursor 16S rRNA levels changed 5-fold versus a 2.5-fold change in total rRNA levels for A. calcoaceticus grown in LB medium. Although the results for the membrane hybridizations and the FISH showed similar trends, the increase in the levels of precursor 16S rRNA and total 16S rRNA was smaller with FISH (3- and <2-fold, respectively [Fig. 4b]) than with membrane hybridizations. A comparison of results obtained by Cangelosi and Brabant (8) with results reported by Licht et al. (17) suggests a similar difference between the results obtained with membrane hybridizations and FISH for measurements made with E. coli. As discussed above, Cangelosi and Brabant (8) used membrane hybridizations to detect a 50-fold increase in the levels of precursor 16S rRNA. In contrast, Licht et al. (17) only reported a ninefold increase in the levels of precursor 16S rRNA using FISH. This apparent discrepancy between the results obtained with membrane hybridizations and FISH may be due to the semiautomated image analysis procedures used to quantify the results of FISH. To quantify fluorescent objects with digital image analysis, a threshold signal intensity must be selected for each data series. This threshold signal intensity represents a balance between including nonspecific hybridization signals (e.g., edge effects) and excluding the hybridization signal of target objects. Therefore, selecting a conservative (e.g., higher) threshold signal intensity excludes some hybridization signal from target objects resulting in a reduction in the magnitude of the change in the levels of precursor 16S rRNA measured with FISH.

Precursor rRNA levels in environmental samples. To test the precursor 16S rRNA probes with environmental samples, we performed membrane hybridizations with RNA extracts from samples of lab- and full-scale activated sludge wastewater treatment systems. Due to the low abundance of Acinetobacter spp. in the examined samples, FISH was not performed. Hybridization results obtained with samples from the sequencing batch reactors appeared to correlate well with the results from the batch growth of A. calcoaceticus in LB medium and filtered wastewater. Since the aerated and unaerated HRTs of the CMAS system and SBR-1 were selected to be comparable, the microbial communities in these two systems experienced similar conditions, with the exception of differences due to continuous versus batch operation. Results suggest that each addition of influent to SBR-1 stimulated rapid growth of Acinetobacter spp. (Fig. 8c), a finding similar to the rapid growth observed after the transfer of the pure culture of A. calcoaceticus into fresh LB medium or filtered wastewater (Fig. 4 and 5). The levels of precursor 16S rRNA in SBR-2 showed similar trends (Fig. 8d).

Conclusion. Our results suggest that quantitative hybridizations with oligonucleotide probes targeting rRNA can be used to measure more than the composition of microbial communities. By combining hybridizations to both precursor 16S rRNA and total 16S rRNA, population size and in situ growth activity can be measured. However, care should be taken in the interpretation of precursor 16S rRNA levels. Already, unexpected effects such as the inhibition of E. coli precursor 16S rRNA processing by unidentified components of mouse intestinal contents have been reported (17). Moreover, our results point at remarkable heterogeneity in the levels of precursor 16S rRNA in Acinetobacter spp. transferred from LB medium to filtered wastewater. Finally, the observation that up to 64% of the total 16S rRNA in A. calcoaceticus existed in the precursor 16S rRNA form suggests that A. calcoaceticus may regulate the production of rRNA (and possibly ribosomes) using a strategy significantly different from that of E. coli. Although the methods presented in this study for measuring in situ growth activity appear promising, more data on rRNA processing and differences in precursor 16S rRNA pools of environmentally important microorganisms are required before this approach can be used widely to determine microbial growth parameters.