TEXT

Top Abstract Text References

Mycobacteria are characterized by long-term survival within vacuoles in the macrophage. There is little detail concerning the supply of nutrients available to the intravacuolar parasite. Peptides might serve as an excellent nutrient source for intracellular mycobacteria. We have undertaken studies with the nonpathogenic species of mycobacteria Mycobacterium smegmatis in order to begin to understand oligopeptide metabolism in the genus. The substrate used was the dipeptide -alanyl-L-histidine, or carnosine. In the present study, a genetic screen was developed to isolate a mutant of M. smegmatis deficient in the utilization of carnosine as a sole source of carbon and nitrogen.

Small peptides can be used as a sole carbon and/or nitrogen source by M. smegmatis (18). To isolate a mutant defective in the utilization of the dipeptide carnosine, the following scheme was devised. First, wild-type cells were plated on basal salts agar (1) with reduced concentrations of glycerol and ammonium chloride as carbon and nitrogen sources, respectively. Very small colonies (<1 mm) were visible on minimal medium agar when glycerol and ammonium chloride were reduced to 0.05 and 0.01%, respectively. When this limiting medium was supplemented with 1.0% carnosine, wild-type cells used carnosine as a source of both carbon and nitrogen and thus produced larger colonies (4 mm). Mutants unable to use carnosine as a carbon or nitrogen source would exhibit small-colony morphology on limiting medium plus carnosine. A population of mutagenized cells (N-methyl-N-nitroso-N-nitroguanidine [1]) was plated on limiting medium plus carnosine and screened for small colony size. A total of 4 × 10⁴ independent colonies were screened. A single mutant colony that showed small-colony morphology on limiting medium agar plus carnosine was recovered.

The mutant strain also grew as small colonies on minimal medium containing limited glycerol and excess ammonium chloride. Mutant colonies, but not wild-type colonies, remained small when carnosine was added to this carbon-limited medium. This demonstrates that there is a defect in the utilization of carnosine as a sole carbon source by the mutant strain. The reciprocal experiment was also performed: mutant cells showed impaired growth compared to wild-type cells when grown in excess glycerol plus carnosine as the sole nitrogen source (data not shown). Thus, the defect in the mutant is common to the two pathways of carnosine utilization as a sole carbon source and a sole nitrogen source. All subsequent experiments were performed in minimal medium containing specific carbon sources and excess ammonium. The strain was named Dpu-1, for dipeptide utilization.

The growth rates of the wild-type (mc²155) and mutant (Dpu-1) strains in minimal glycerol and minimal carnosine were measured and compared. Table 1 shows that mc²155 and Dpu-1 had comparable growth rates (doubling time, 4 h) in minimal glycerol medium. However, the wild-type strain grew more slowly in minimal carnosine medium than on glycerol (doubling time, 20 h). The mutant grew even more slowly (doubling time, 42 h). This suggests that the metabolic defect exhibited by the mutant is specific for peptide utilization.

There are at least three explanations for failure to use a given peptide as a sole carbon source: defective transport of the substrate, impaired hydrolysis of the substrate, or failure of subsequent catabolism of its constituent amino acids. To distinguish among these, we examined the growth of wild-type and mutant cells in liquid minimal medium on several amino acids and small peptides as sole carbon sources. Table 1 shows the growth rates of the wild-type and mutant strains in medium containing one or both of the two constituent amino acids of carnosine, -alanine (-Ala) and L-His. The wild-type and mutant strains showed comparable growth on the two amino acids, -Ala and L-His, supplied alone or together. Thus, the mutant was not impaired in its ability to catabolize the amino acid components of the dipeptide carnosine. In addition, this result demonstrates that the metabolism of one component did not interfere with the metabolism of the other in the mutant strain. Finally, the mutant utilized other, structurally related (L-Ala and L-Asp) or unrelated (L-Pro) amino acids with an efficiency comparable to that of the wild type (data not shown).

The M. smegmatis mutant described here used a number of related and unrelated peptides as sole carbon sources with the same degree of efficiency as the wild-type parent. Growth on a range of dipeptides, including those with -Ala or L-His in either the N- or the C-terminal position, was examined in wild-type and mutant cells. The dipeptides -Ala-L-His (carnosine) and L-Arg--Ala were efficiently utilized by the wild type and not by the mutant. The dipeptides -Ala-L-Ala and -Ala-Gly, bearing -Ala in the N-terminal position, were not utilized efficiently by either the wild type or the mutant. At such slow growth rates (doubling times of 50 and 132 h), there was no detectable difference between the wild type and the mutant. Uptake of one of these poor growth substrates (-Ala-L-Ala) was shown to be inefficient in both the wild type and the mutant (data not shown). In contrast, dipeptides containing L-His in either position were utilized at comparable efficiencies by both strains. Finally, wild-type and mutant cells showed comparable growth rates on all tripeptides tested (data not shown).

Taken together, these observations suggest that M. smegmatis has peptide permeases of differing specificities. The -Ala-containing dipeptides associated with basic amino acids (L-Arg and L-His) were used relatively efficiently for growth and may be recognized and transported by the same permease. On the other hand, -Ala-containing dipeptides with small, neutral C-terminal amino acids were not efficiently utilized by M. smegmatis.

The transport of dipeptides into bacteria is mediated by a peptide permease(s). The standard method of measuring transport relies on accumulation of a radiolabeled substrate by the cells. The level of radiolabel within the cells reflects the amount of the substrate accumulated over time. However, radiolabeled peptides are not readily available, and an alternative method was sought to evaluate the transport of carnosine by wild-type and Dpu-1 cells. Payne and colleagues pioneered a method for the evaluation of peptide transport (15). In their assay, the amount of a substrate taken up by a cell suspension is measured by labeling the supernatant of the cells with the fluorescent peptide-labeling chemical dansyl chloride. The method used here is a modification of this technique; the peptide concentration remaining in the supernatant is measured by the absorbance at 212 nm.

Figure 1A shows the rates of carnosine removal from the supernatants of wild-type and Dpu-1 cells. The initial rates of peptide removal from the supernatant by the mutant strain were significantly lower than those exhibited by the wild-type strain. Thus, there is a clear defect in the ability of the mutant strain to remove carnosine from the culture supernatant. In contrast, wild-type and mutant cells were equally capable of removing the dipeptide L-Ala-L-His from the supernatant (Fig. 1B). Note, however, that the Dpu-1 mutant still grew, albeit very slowly, on carnosine as the sole carbon source (Table 1), suggesting that an alternate means of dipeptide uptake is retained by the mutant.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Removal of the dipeptide carnosine (A) and L-Ala-L-His (B) from the supernatant by wild-type mc2155 (open squares) and mutant Dpu-1 (solid diamonds) cells. Cells in the mid-logarithmic-growth phase were washed, concentrated to an OD600 of 1.0, and resuspended in minimal medium (basal salts [1]). At t = 0, 100 µM substrate was added. Aliquots were collected at the times indicated, and the supernatant was filtered away from the cells with 0.22-µm-pore-size filters (Millipore). The peptide concentrations of the samples were measured at 212 nm. At each time, the A212 values in simultaneous cultures without added substrate were subtracted from the experimental values. These control values ranged from 0.01 to 0.08. Data were expressed as nanomoles of peptide remaining per milliliter of supernatant and were derived from four data sets.

Mycobacterial cells are characterized by a complex cell wall and a hydrophobic cell surface (9, 12, 13). In the uptake assays used here, the sticking of peptides to the cell surface would simulate their uptake into the cell. To rule out this possibility, peptide uptake assays were performed at 4°C, and no change in the peptide concentrations in the supernatants of cells was observed (data not shown).

Measurement of transport is often confounded by the presence of subsequent utilization steps. An observed defect in uptake may reflect the mutant's inability to metabolize the dipeptide subsequent to transport. Once peptides enter the cell, they are cleaved to constituent amino acids. L-His will be catabolized by enzymes similar to those of the hut systems found in Bacillus subtilis (3, 4), Salmonella typhimurium (10), and Aerobacter aerogenes (7). -Ala metabolism has not previously been reported in mycobacteria, but it has been studied in E. coli, where it is a precursor in the pantothenate synthesis pathway (2).

To determine whether the lower levels of carnosine accumulation by the mutant strain were the result of impaired peptidase activity, carnosinase activity was measured in wild-type and mutant cells. Carnosine is cleaved by specific peptidases in other bacteria, namely, PepV in Lactococcus delbrueckii (19) and PepD in S. typhimurium (5). Intra- and extracellular peptidase activities have been identified in Mycobacterium phlei (16, 17). Extracts prepared from mid-log-phase cultures of wild-type and mutant cells were incubated in the presence of 4.4 mM carnosine. The appearance of free L-His in the reaction was measured by o-phthalaldehyde labeling and detected fluorimetrically. The carnosinase levels in the wild-type and mutant strains were measured as 1.17 ± 0.09 and 1.14 ± 0.12 nmol of carnosine cleaved per mg of protein per min, respectively. Heat-inactivated extracts were incubated with carnosine, and no released histidine was detectable.

The method used here to evaluate carnosine uptake relies on the detection of the change in the extracellular concentration of peptide bonds. The possibility remained that an extracellular peptidase was cleaving the carnosine in the supernatant of wild-type cells. The resulting amino acids would then be transported into the cell by their respective permeases. To detect extracellular cleavage of carnosine, cell suspensions were exposed to 100 µM carnosine and the supernatants were collected at 0 and 20 min. The supernatants were concentrated, and free amino acids (i.e., L-His) were labeled with o-phthalaldehyde. High-pressure liquid chromatography (HPLC) was used to detect derivatized histidine in the supernatant. In a control experiment, the concentrations in the supernatant predicted by the uptake assays, in which 10% of the substrate is removed from the supernatant over 20 min, were reconstructed. Thus, a solution of 90 µM carnosine and 10 µM L-His was labeled and analyzed by HPLC. This method has sufficient sensitivity to detect L-His at 10 µM. Measured at 0 and 20 min, the supernatants of wild-type cells contained no detectable L-His, indicating that carnosine is not cleaved extracellularly by wild-type M. smegmatis (data not shown).

In conclusion, a mutant of M. smegmatis defective in the utilization of the dipeptide carnosine for growth has been isolated and found to be defective in carnosine transport. Carnosine uptake rates are clearly inhibited in the mutant strain compared to those in the wild type. However, measurement of uptake rates can be confounded by the immediate incorporation and trapping of the substrate in the next step of its metabolic pathway. Thus, analysis of subsequent metabolic steps is crucial to the physiological characterization of transport mutants. Here, the predicted peptide cleavage step following the uptake of carnosine has been measured in wild-type and mutant strains and found to be identical. Furthermore, no carnosine cleavage was detected in the supernatant of either strain.

The uptake rates for carnosine exhibited by the wild-type strain were estimated from the data in Fig. 1A. To make these calculations, it was assumed that a culture at 0.3 mg/ml (dry weight) results in an optical density at 600 nm (OD₆₀₀) of 0.8 (6). The cell volume is estimated to be 3 µl/mg (dry weight) (11). The rate of uptake of carnosine by wild-type M. smegmatis was approximately 2 nmol/mg/min. This is well within the range recorded for oligopeptide transport by several different organisms (8, 14, 15). The identities and substrate specificities of the dipeptide permeases will be established by our ongoing detailed analysis of the uptakes of a large range of peptides and by genetic studies.