JOURNAL OF BACTERIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Feb. 2000, p. 919–927 Vol. 182, No. 4
Amino Acid Transport and Metabolism in Mycobacteria: Cloning,
Interruption, and Characterization of an L-Arginine/?-Aminobutyric
Acid Permease in Mycobacterium bovis BCG
ANJALI SETH AND NANCY D. CONNELL*
Department of Microbiology and Molecular Genetics and New Jersey Medical School National Tuberculosis Center,
UMDNJ/New Jersey Medical School, Newark, New Jersey 17103
Received 22 June 1999/Accepted 19 November 1999
Genes encoding L-arginine biosynthetic and transport proteins have been shown in a number of pathogenic
organisms to be important for metabolism within the host. In this study we describe the cloning of a gene
(Rv0522) encoding an amino acid transporter from Mycobacterium bovis BCG and the effects of its deletion on
L-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using
primers homologous to the rocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant
strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker.
The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains
was conducted using14C-labeled substrates. Greatly reduced uptake of L-arginine and ?-aminobutyric acid
(GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild
type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a
minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow), L-[14C]argi-
nine uptake by the mutant but not the wild-type strain increased strongly. Exogenous L-arginine but not GABA,
lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells
growing in optimal carbon and nitrogen sources such as glycerol and ammonium. L-Arginine supplied in the
form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain
showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.
The mycobacteria are distinguishable from most other or-
ganisms on the basis of their low permeability. Cell wall struc-
tural analysis (41) and permeability studies (18, 28) have
provided an increased understanding of this characteristic.
However, few of the mycobacterial transporters mediating the
uptake of nutrients have been characterized at the genetic,
molecular, or biochemical levels. There are few recent studies
of nutrient transport by mycobacteria (9; for a review, see
reference 18), and, in particular, there are no studies of the
role of nutrient transport in the intracellular survival of myco-
bacteria. The nature and availability of carbon, nitrogen, and
energy sources within the macrophage can best be studied with
genetic mutants altered in intermediary metabolism and trans-
port. This study presents a genetic approach to the transport of
L-arginine in mycobacteria.
Many microorganisms use L-arginine as a source of energy
and/or nitrogen, and the pathways of L-arginine biosynthesis
and utilization are well understood in some cases. At least five
pathways of L-arginine catabolism have been identified in mi-
croorganisms, listed here by the first enzyme to act upon the
substrate: arginase, arginine deiminase, arginine decarboxyl-
ase, arginine succinyl transferase, and arginine oxidase. L-Ar-
ginine metabolism has not previously been examined in the
slow-growing mycobacteria, but scrutiny of the genomic se-
quence of Mycobacterium tuberculosis (16) reveals the presence
of two of these five enzymes, arginine deiminase (Rv1001) and
arginine decarboxylase (Rv2531c). Arginine decarboxylase has
been studied in the context of regulation of pyrimidine synthe-
sis in M. smegmatis (2, 3, 5, 40).
In the arginase pathway, L-arginine is converted to urea and
ornithine. Urea is subsequently converted to NH3and CO2by
urease. There is no apparent homolog of arginase in the
Sanger database, but homologs of genes of subsequent en-
zymes in the arginase pathway are present in the H37Rv
genome. In addition, the urease genes of BCG and M. tuber-
culosis have been cloned and characterized (15, 46). By con-
structing a strain lacking the urease gene, it was demonstrated
that ureolytic activity was not essential to BCG ?ure grown in
vitro. However, a slight decrease in the multiplication and
persistence of the mutated strain compared with wild-type
BCG was observed in the lungs of infected mice (47).
L-Arginine transport is an important aspect of arginine me-
tabolism and is regulated in concert with L-arginine catabolic
enzymes in many bacterial systems. Several different classes of
permeases are responsible for L-arginine transport among the
bacteria: the existence within a single organism of multiple
transport systems for this amino acid attest to its importance.
In Escherichia coli and Salmonella, the major L-arginine per-
mease is a member of the binding protein-dependent family of
transporter systems, with three separate periplasmic binding
proteins of differing specificities (24). The first binds L-lysine,
L-arginine, and L-ornithine (11, 48); the second binds L-argi-
nine and L-ornithine (13); and the third binds only L-arginine
In Bacillus subtilis, the rocE and rocC genes encode putative
arginine permeases and are homologous to each other (23, 42).
The sequence of these permease genes probably classifies them
as members of the amino acid-polyamine-organocation super-
family, single-protein membrane carriers characterized by 12
or 14 transmembrane ?-helices (37). To study L-arginine trans-
* Corresponding author. Mailing address: Department of Microbi-
ology and Molecular Genetics, UMDNJ/New Jersey Medical School,
185 South Orange Ave., Newark, NJ 07103. Phone: (973) 972-3759.
Fax: (973) 972-3644. E-mail: email@example.com.
port in mycobacteria, the M. tuberculosis sequence was
searched for predicted open reading frames (ORFs) with ho-
mology to the B. subtilis L-arginine transport proteins. Two
homologs were identified, Rv0522 and Rv2320c.
Here, Rv0522 was cloned from BCG and used to construct
a strain lacking the permease encoded by this ORF. The de-
letion strain and its wild-type parent are characterized with
respect to transport, growth properties, and survival in a mu-
rine macrophage cell line.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The bacterial strains and
plasmids used in this study are described in Table 1. Escherichia coli strains were
grown in Luria-Bertani medium, and mycobacterial strains were grown in
Middlebrook medium [per liter, (NH4)SO4, 0.5 g; L-glutamic acid, 0.5 g; sodium
citrate, 0.1 g; pyridoxine, 0.001 g; biotin, 0.0005 g; Na2HPO4, 2.5 g; KH2PO4,
1.0 g; ferric ammonium citrate, 0.04 g; MgSO4, 0.05 g; CaCl2, 0.0005; ZnSO4,
0.001; CuSO4, 0.001 g]. Middlebrook 7H9 (liquid) and 7H11 (1.5% agar) media
(Difco) were supplemented with glycerol (0.5% vol/vol) and ADC supplement
(0.5% bovine serum albumin, fraction V [Boehringer Mannheim], 0.2% dex-
trose, 0.85% NaCl). Antibiotics were added at the following concentrations:
ampicillin, 100 ?g/ml, kanamycin and streptomycin, 50 ?g/ml for E. coli and 20
?g/ml for BCG; and hygromycin, 150 ?g/ml for E. coli and 50 ?g/ml for BCG.
The minimal medium used was composed of basal salts (0.1% KH2PO4, 0.25%
NaH2PO4, 0.5% NH4Cl, 0.2% K2SO4) medium supplemented with glycerol
(0.5%). Sauton’s medium was used without amino acid supplements (17). Where
applicable, the nitrogen (NH4Cl) and/or carbon (glycerol and/or dextrose)
sources were omitted. All liquid cultures of BCG were supplemented with 0.05%
Tween 80 (Sigma Chemicals).
Cloning and DNA manipulations. Plasmid DNA preparations, restriction en-
donuclease digestions, alkaline phosphatase treatments, ligations, transforma-
tions, and other DNA manipulations were performed by standard procedures
(36). For electroporation of BCG, the cells were grown until the culture reached
a turbidity (optical density at 600 nm [OD600]) of about 0.6 and then harvested
at 4,000 ? g for 10 min. The pellet was washed with 10% glycerol and centrifuged
again at 4,000 ? g for 10 min. The procedure was repeated two more times
before resuspension of the pellet in 1/10 volume of 10% glycerol. All manipu-
lations were carried out at 37°C. DNA (3 to 5 ?g) was added to 0.5 ml of cells
in a 0.4-cm electroporation cuvette (BTX). The cuvette was subjected to a single
pulse using the Bio-Rad Gene Pulser set at 2.5 kV and 25 ?F, with the pulse
controller resistance set at 1,000. The contents of the cuvette were diluted into
5 ml of 7H9 medium and incubated overnight at 37°C. After incubation, the
entire 5 ml was centrifuged and plated onto 7H10-antibiotic plates. Transfor-
mants appeared after incubation at 37°C for 3 to 4 weeks.
Construction of BCG Rv052 deletion strain. PCR analysis to screen the BCG
genomic library to isolate a cosmid containing the B. subtilis rocE homolog was
performed using the PCR kit from Boehringer Mannheim Biochemicals. The
primers used were 5? ATCGTGATCTTCTTCGTCGG 3? and 5? ATGATCAC
GCACAGGAATCC 3?. The cosmid DNA was digested with a number of re-
strictions enzymes, and a Southern analysis using the PCR product as a probe
revealed the presence of a 6-kb EcoRI fragment bearing the homolog. This
fragment was then subcloned into pGEM3Zf? and is called pAS5 (see Fig. 1).
This was then transformed into a dam E. coli strain, GM48. Transformation into
GM48 permitted digestion with the enzyme BclI, which released a 191-bp frag-
ment. A Kan-Str cassette as a BamHI fragment from plasmid pSM240 was then
inserted into the BclI site of pAS5 to create pAS6. This construct was then used
for allelic exchange of the homolog, the Rv052 gene in BCG.
PCR and Southern analysis were used to differentiate between single- and
double-crossover events (see Fig. 1). BCG genomic DNA was isolated using
N-acetyl-N,N,N-trimethylammonium bromide) (Sigma) as previously described
(18). PCR was performed using the Expand Long Template Kit (Boehringer
Mannheim) as specified by the manufacturer. The primers 5? AGCCACATCC
GTACCCCCAG 3? and 5? CACGCATCGTGATCTTCTTCG 3? flank the Kan-
Str cassette such that single crossovers would show fragments of 471 bp of the
wild-type copy of the gene and the 3.5-kb fragment containing the Kan-Str
marker. True allele replacements would lack the small band (deleted by recom-
bination out of the genome) and would show only the 3.5-kb fragment. The PCR
results were confirmed by Southern analysis of the double recombinant with the
Kan-Str marker as a probe. Southern hybridization was performed by the method
of Maniatis et al. (36). The Southern analysis also shows that the Kan-Str cassette
inserted only once in the genome.
Uptake assays. Cells were grown to mid-log phase, washed three times with
basal salts medium plus Tween 80 at 0.05%, and concentrated in basal salts
medium approximately fivefold to a final OD600of 3.0. The cell suspensions (1
ml) were warmed to 37°C with shaking. The culture was treated with rifampin
(200 ?g/ml) for 10 min prior to initiation of an uptake assay to block transcrip-
tion and subsequently protein synthesis; this prevented the unlimited incorpo-
ration of radiolabeled amino acids into protein. The uptake reaction was initi-
ated by the addition of radiolabeled substrate plus unlabeled substrate at the
specific activities (usually 40 to 50 ?Ci/?mol) and to the final concentrations
(usually 100 ?M) described in the legends of the figures. Incorporation was
terminated by removal of 0.1-ml samples at the indicated time to filters (What-
man GF/F; 0.45 ?m) prewetted with BS medium. The cells were rinsed quickly
(within 10 to 15 s) three times with 7 ml of ice-cold basal salts medium plus
Tween 80 on a Hoeffer 10-place manifold with air vacuum. Filters with cells
thereon were transferred to vials containing 5 ml of scintillation fluid for deter-
mination of radioactivity. The counts per minute were normalized to milligrams
of protein per 0.1-ml aliquot for each cell suspension; protein was determined by
the Bio-Rad protein assay.
For analysis of uptake by cells resuspended in different media, care was taken
to ensure that the cultures used for uptake studies were at similar densities
before the washing and concentration steps. This was essential when cells were
resuspended in a medium that does not support measurable growth (minimal
salts plus amino acid or ?-aminobutyric acid [GABA]) and compared with cells
resuspended in Middlebrook medium.
To measure the effect of exogenous L-arginine on [3H]uracil incorporation, 0.5
?Ci of [3H]uracil (38.5 Ci/mmol) (New England Nuclear) and peptide were
added simultaneously to the cells to initiate the experiment. After 16 h, the
cultures were precipitated in 10% trichloroacetic acid (TCA) (Sigma) at 4°C for
TABLE 1. Strains and plasmids used in this study
Designation Relevant characteristics
E. coli strains
supE44 ?lacU169 (?80lacZ?M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
dam-3 dcm-6 thr-1 ara-14 tonA31 lacY1 tsx-78 glnV44 galK2 galT22 thi-1
M. bovis BCG strains
BCG (Rv0522 interrupted with Kan-Str)
AS1 complemented with a wild-type copy of Rv0522 (pAS7)
Ampr; multiple-cloning-site vector
Kanr; E. coli-mycobacterium integrating shuttle vector
Kan-Str cassette in pBEN
Kanr; E. coli-mycobacterium shuttle expression vector with hsp60 promoter
pUC19 containing Hygrfrom pIJ963 as a SpeI fragment
pGEM3Zf? with a 6-kb EcoRI fragment from cosmid 121 of the pYUB18 BCG cosmid library
pAS5 with a 3.5-kb BamHI Kan-Str cassette from pSM240 in the BclI site of pAS5
pMV305 with the 6-kb EcoRI fragment from pAS5 and a HygrSpeI fragment from pJB3 in the
Kanr; cosmid library vector
920SETH AND CONNELLJ. BACTERIOL.
20 min, filtered, and washed three times with 5 ml of cold 10% TCA over filters
(Whatman GF/F; 0.45-?m pore size) prewetted with 10% TCA on a Hoeffer
10-place manifold with air vacuum (as above).
Macrophage survival assays. J774.1 cells were maintained in Dulbecco’s mod-
ified Eagle’s medium (Sigma) supplemented with 10% fetal calf serum (Sigma),
amino acids (BioWhittaker), and L-glutamine (Sigma). The cells were subcul-
tured into 96-well plates (1.5 ? 105cells per well) for 6 to 12 h. Then 2 ? 106
CFU of logarithmically growing bacteria were washed in macrophage medium
and placed in the wells. After 4 to 6 h of attachment, the wells were rinsed three
times in macrophage medium. The numbers of intracellular bacteria was deter-
mined by removing the supernatant of three wells in parallel, lysing the macro-
phages in double-distilled H2O ddH2O, diluting, and plating for CFU on 7H11
plates with the appropriate antibiotics. The supernatants were checked for the
presence of extracellular bacteria at every time point, and the number of extra-
cellular bacteria remained in the range of 1 to 5% of that of intracellular
organisms. Triplicate determinations were made for each time point, and the
experiment was performed five times.
Cloning and interruption of the rocE permease homolog
from BCG. The rocE gene of B. subtilis encodes a major L-
arginine permease. Its expression is induced by L-arginine in
the medium, controlled at the level of transcription by the
product of the rocR gene, a member of the NtrC/NifA family of
regulators. Using the amino acid sequence of the B. subtilis
rocE gene, the Sanger TB database was analyzed for ORFs
with homology to the rocE gene. The strongest homology was
exhibited by Rv0522 (31% identity and 53% similarity). The
sequence of this predicted ORF in turn shows 44.3% identity
to the GABA permease gene (gabP) of E. coli and 20% identity
to gabP of B. subtilis. In B. subtilis, the rocE gene is part of the
rocDEF operon bearing the genes for L-arginine catabolism. In
contrast, Rv0522 is not part of an operon and is flanked by
divergent ORFs of unknown function.
Primers derived from the Sanger database (16) were used to
screen a genomic library of BCG (Pasteur) DNA in pYUB18
(27) for the presence of Rv0522 sequences. A cosmid carrying
the BCG homolog of Rv0522 was identified (Fig. 1). From this
cosmid, an EcoRI fragment of 6 kb bearing the BCG Rv0522
homolog was cloned into pGEM3Zf to create pAS5 (Table 1).
A 191-bp BclI fragment in the ORF was removed and replaced
with an antibiotic cassette containing kanamycin and strepto-
mycin resistance markers to create pAS6. This final construct
was linearized with BamHI and electroporated into BCG.
Transformants resistant to both kanamycin and streptomycin
were selected. The candidates for allele replacements were
first screened by PCR, using oligonucleotide primers flanking
the marker insertion site (see Materials and Methods). South-
ern blot analysis confirmed the structure of genomic DNA
representing the wild-type strain and the allele replacement
(double crossover) strain, AS1 (Fig. 1).
To complement the deletion, a wild-type copy of the Rv0522
gene was inserted in single copy into AS1. The mycobacteri-
ophage L5 attachment site (attB) was used for integration of
pAS7, the pMV305 plasmid carrying the attP site of L5 (32)
and a wild-type copy of the BCG Rv0522 gene. The comple-
mented mutant strain was named AS2.
Uptake properties of AS1 (?Rv0522). AS1 and its wild-type
parent were analyzed for uptake of various L-amino acids by
using radiolabelled substrates. The cells were grown in Middle-
brook medium (7H9 plus glycerol, ADC supplement, and
0.05% Tween). The mutant strain showed a significant de-
crease in uptake of L-[14C]arginine (90% reduction) (Fig. 2A).
In addition, the mutant showed a clear defect (75% reduction)
in uptake of [14C]GABA compared with wild-type cells (Fig.
2B). Neither L-[14C]arginine nor [14C]GABA uptake was com-
pletely abolished. This was not unexpected, as most bacteria
have multiple transporters for L-arginine. There was no differ-
ence between the wild type and mutant in uptake of the struc-
turally related amino acids
L-[14C]lysine (Fig. 2D). The complemented mutant strain, AS2,
contained a wild-type copy of the Rv0522 gene supplied in
trans that fully complemented the L-[14C]arginine (Fig. 2A)
and [14C]GABA (data not shown) uptake defects. Comparison
of the uptake of structurally unrelated amino acids, L-alanine
and L-proline, indicated that there were no differences between
the wild-type and AS1 strains (data not shown). Finally, the
apparent Kms for transport of L-[14C]arginine and [14C]GABA
were calculated from Lineweaver-Burk analyses (Fig. 3) and
found to be 250 and 165 ?M, respectively.
L-[14C]arginine uptake by wild-type BCG and AS1. In other
bacteria, expression of L-arginine catabolic and biosynthetic
operons is regulated by the presence or absence of exogenous
L-arginine (7, 13, 23, 53). This regulation is often mediated by
L-arginine acting as a corepressor in concert with the ArgR/
AhrC protein as a repressor (34). To examine the effect of
exogenous L-arginine on uptake of L-[14C]arginine in wild-type
BCG, cells in balanced growth in Middlebrook medium were
washed and incubated in minimal salts medium (see Materials
and Methods) with 20 mM L-arginine as the sole carbon and
nitrogen source. After 24 h at 37°C, uptake of L-[14C]arginine
by cells incubated in L-arginine alone was only slightly reduced
(25%) in comparison to that by wild-type cells growing in
Middlebrook medium (Fig. 4A). The effect of starvation for
carbon and nitrogen on L-[14C]arginine uptake by wild-type
BCG was also examined. After 2 h (data not shown) or 24 h
(Fig. 4A) of starvation in basal salts lacking sources of carbon
and nitrogen, L-[14C]arginine uptake by wild-type cells was
Mid-log-phase AS1 cells were washed and incubated for 2 h
(data not shown) or 24 h in minimal salts with 20 mM arginine.
Figure 4B shows that in contrast to the reduction seen in
wild-type BCG, L-[14C]arginine uptake by the AS1 mutant was
L-[14C]ornithine (Fig. 2C) or
FIG. 1. Construction of Rv052 deletion strain. (A) Cloning strategy for con-
struction of AS1. See Materials and Methods for details. (B) Southern analysis
of genomic DNA from BCG and AS1 strains. Genomic DNA from a single-
crossover recombinant (lane 1), wild-type BCG (lane 2), and mutant AS1 (lane
3) and plasmid DNA from pAS6 (lane 4) were digested with EcoRI and probed
with the Kan-Str casette.
VOL. 182, 2000AMINO ACID PERMEASE OF BCG 921
strongly increased over the levels found in AS1 grown in 7H9
medium. The results were the same after 2 h in 20 mM argi-
nine. This suggests that exogenous L-arginine in the medium
may induce a permease other than that encoded by Rv0522.
Uptake of L-[14C]arginine by starved AS1 cells was also
measured (Fig. 4B). Surprisingly, under these conditions, the
mutant strain showed levels of uptake higher than under any
other conditions, including those exhibited by the wild type
after growth on Middlebrook medium.
[14C]GABA uptake by wild-type BCG and AS1. To examine
the effect of exogenous GABA on [14C]GABA uptake in BCG,
wild-type cells in balanced growth in Middlebrook medium and
cells washed and incubated in minimal salts medium with 20
mM GABA as the sole carbon and nitrogen source were com-
pared. Figure 5A shows that there was a 70% reduction in
[14C]GABA uptake by wild-type cells after exposure to 20 mM
GABA. The results were identical after only 2 h of incubation
in 20 mM GABA (data not shown).
The relative contribution of the Rv0522 permease to this
reduction in GABA uptake was evaluated by measuring the
effect of GABA exposure on [14C]GABA uptake by AS1 cells.
Figure 5B shows that uptake of [14C]GABA was reduced 60%
in AS1 after 24 h of incubation in 20 mM GABA, in compar-
ison with mutant cells in Middlebrook medium.
Cultures growing in Middlebrook medium were washed and
incubated in minimal salts with no carbon or nitrogen source.
The effects of this starvation on [14C]GABA uptake by wild-
type BCG were evaluated and shown in Fig. 5A. There was a
70% reduction in uptake of [14C]GABA by wild-type cells after
starvation, similar to that exhibited by wild-type cells after
GABA exposure. AS1 cells also showed a reduction compara-
ble to that exhibited after GABA exposure (Fig. 5B). Thus,
unlike GABA uptake in B. subtilis (21), GABA uptake by
wild-type BCG was not induced by starvation for carbon and
nitrogen, and was, in fact, slightly repressed. Furthermore, the
reduction shown in [14C]GABA uptake by AS1 suggested that
a wild-type uptake activity not affected by the deletion of
Rv0522 was responsive to carbon and nitrogen starvation.
L-Arginine utilization by BCG. Growth of wild-type BCG
and AS1 cells in minimal arginine medium was measured. The
FIG. 2. Uptake of14C-labeled amino acids by wild-type BCG (?), AS1 (Œ), and AS2 (E). (A) Uptake of L-[14C]arginine. (B) Uptake of [14C]GABA. (C) Uptake
of L-[14C]ornithine. (D) Uptake of L-[14C]lysine.
922 SETH AND CONNELLJ. BACTERIOL.
cells were first grown to the mid-logarithmic growth phase in
standard Middlebrook medium. They were washed and resus-
pended at an OD600of 0.2 in various minimal media (basal
salts medium or Sauton’s medium [see Materials and Meth-
ods]) containing at 1 or 20 mM L-arginine as the sole carbon
and/or sole nitrogen source. Surprisingly, neither wild-type nor
mutant cells were capable of growing in either medium with
L-arginine as the sole carbon or nitrogen source. In basal salts
medium or Sauton’s medium supplemented with glycerol
(0.5%) and ammonium chloride (0.5%), wild-type cells grew at
rates comparable to those seen for Middlebrook 7H9-grown
cells. The following amino acids were tested as sources of
either carbon or nitrogen to support the growth of wild-type
BCG: L-histidine, L-lysine, L-ornithine, GABA, L-alanine, and
L-proline. In all cases, single amino acids were incapable of
supporting growth. This is in marked contrast to the observa-
tion that a wide range of amino acids and di- and tripeptides at
a concentration of 2 to 5 mM are capable of supporting the
growth of wild-type M. smegmatis in minimal medium as either
the sole carbon or nitrogen source (9, 45).
Effect of exogenous arginine on wild-type BCG. Wild-type
BCG was grown in Middlebrook 7H9 medium, containing glyc-
FIG. 3. Lineweaver-Burk analysis of L-arginine and GABA uptake. Double inverse plots of [14C]GABA (A) and L-[14C]arginine (B) are shown. Uptake proceeded
for 2 min at the concentrations shown on the x axis. The apparent Kms were calculated from the x axis intercept. 1/s ? (nanomoles of substrate/milligram of
protein/minute)?1; 1/v ? (micromoles of substrate)?1.
FIG. 4. Comparison of uptake of L-[14C]arginine by BCG and AS1 under different nutritional conditions. Uptake of L-[14C]arginine by cells growing in Middlebrook
medium (I), after overnight starvation (no carbon and nitrogen source) (?), or after overnight treatment with 20 mM L-arginine as the sole carbon and nitrogen source
(Œ) is shown. (A) Uptake by wild-type BCG. (B) Uptake by AS1.
VOL. 182, 2000 AMINO ACID PERMEASE OF BCG923
erol and ammonium, in the presence of exogenous L-arginine
at concentrations ranging from 0 to 40 mM (Fig. 6). Note that
in this experiment, the primary sources of carbon and nitrogen
(glycerol and ammonium, respectively) provided by Middle-
brook medium are optimal for growth of the slow-growing
mycobacteria (45). Surprisingly, at 15 mM arginine, there was
some inhibition of growth, and at 20 mM, growth was com-
pletely inhibited. Structurally related amino substrates, such as
L-ornithine (Fig. 6), L-lysine (results not shown), and GABA
(results not shown), and unrelated amino acids (L-proline and
L-alanine [results not shown]) at the same concentrations
showed no inhibition of growth; therefore, this growth inhibi-
tion was specific for arginine. Furthermore, growth inhibition
by L-arginine was reduced in AS1 (Fig. 6), as would be pre-
dicted from the reduced arginine transport in AS1 cells (Fig.
Amino acids can also be supplied to cells in the form of
peptides. Indeed, Marquis et al. showed that in culture, thre-
onine auxotrophs of Listeria monocytogenes grow poorly on
free threonine and quite well on threonine-containing pep-
tides. These same auxotrophs showed no difference in growth
rate within threonine-starved J774 macrophages, suggesting
that threonine-containing peptides are available for intracyto-
plasmic growth (39). In BCG, there is no toxicity of exogenous
L-arginine when supplied at 20 or 30 mM in the form of
High concentrations of exogenous L-arginine are cytocidal.
To determine whether the effect of exogenous arginine on the
growth of wild-type BCG is cytostatic or cytocidal, the cultures
described in Fig. 6 were plated to determine CFUs. There was
no effect of 10 mM arginine on viability, but the culture con-
taining 20 mM arginine contained no viable cells (data not
shown). To confirm this observation, a labeling assay (14) was
used to evaluate the metabolic activity of wild-type BCG ex-
posed to exogenous arginine. [3H]uracil incorporation into
precipitable macromolecules was inhibited by 50% in wild-type
BCG growing in rich medium and incubated in 20 mM L-
arginine for 24 h (data not shown). In contrast, the addition of
20 mM L-arginine to BCG resuspended in basal salts medium
(starvation conditions) had little effect (10% inhibition) on
[3H]uracil incorporation. Thus, the effect of exogenous L-argi-
L-arginyl-L-asparagine (data not
FIG. 5. Comparison of uptake of [14C]GABA by BCG and AS1 under different nutritional conditions. Uptake of [14C]GABA by cells growing in Middlebrook
medium (I), after overnight starvation (no carbon or nitrogen source) (?), or after overnight treatment with 20 mM GABA as the sole carbon and nitrogen source
(}) is shown. (A) Uptake by wild-type BCG. (B) Uptake by AS1.
FIG. 6. Effect of exogenous L-arginine or L-ornithine on growth of wild-type
and mutant BCG. Exponentially growing cells were washed and resuspended at
an OD600of 0.1 in Middlebrook 7H9 medium in the presence of exogenous
L-arginine at the concentrations indicated. The OD600was measured after 4 days
of growth at 37°C. From left to right for each concentration, wild-type BCG plus
L-arginine (open bars), AS1 plus L-arginine (solid bars), wild-type BCG plus
L-ornithine (hatched bars), and AS1 plus L-ornithine (shaded bars). The exper-
iment is representative of three similar experiments.
924SETH AND CONNELLJ. BACTERIOL.
nine on [3H]uracil incorporation appears to depend on the
nutritional state of the culture.
Survival of strain AS1 in cultured murine macrophages.
Roles for genes involved in L-arginine transport (30) and me-
tabolism (35) have been implicated in macrophage infection
studies (see Discussion). Wild-type BCG, AS1, and AS2 were
evaluated for survival in unactivated J774.1 macrophages. No
differences were found among the three strains.
Little is known about the regulation of amino acid transport
and metabolism in mycobacteria. As a first step to understand-
ing L-arginine metabolism in BCG, we screened the Sanger
database for ORFs with homology to the arginine permeases
of E. coli, Pseudomonas spp., and B. subtilis. The highest ho-
mology pointed to Rv0522, which was homologous to the rocE
arginine permease of B. subtilis. Rv0522 also showed 20%
identity and 19% similarity to the B. subtilis gabP gene. The
BCG homolog of Rv0522 was cloned, interrupted, and crossed
onto the chromosome of BCG by gene replacement. The mu-
tant thus constructed, AS1, showed decreased uptake of both
L-[14C]arginine and [14C]GABA.
Arginine metabolism has been widely studied in bacteria
(see the introduction), and a range of regulatory systems con-
trols the arginine catabolic genes, including those encoding
transporters. In the enterics, L-arginine uptake systems are
either repressed or unaffected by exogenous L-arginine: the
ArgR protein functions largely in the repression of the arginine
biosynthetic operons (34). In P. aeruginosa and B. subtilis, ex-
ogenous L-arginine induces L-arginine uptake (7, 43).
There are no published reports of L-arginine or GABA
transport by the slow-growing mycobacteria. L-arginine trans-
port by the fast-growing species M. phlei has been measured as
part of a larger study of the energetics of amino acid transport
(45, 54). From our data, we estimate initial uptake rates of
L-arginine in the range of only 0.014 nmol/mg/min, which is
10-fold lower than those measured in E. coli (48), P. aeruginosa
(52), and Clostridium (50). In B. subtilis, the Kmfor GABA
transport is 37 ?M (10). The apparent Kms measured here
(L-arginine, 250 ?M; GABA, 165 ?M) are significantly higher
than those described in other systems. The absolute levels of
uptake of amino acids described in the present study point to
low uptake levels as one possible impediment to utilization of
the substrates for growth. In Salmonella enterica serovar Ty-
phimurium for example, L-arginine transport severely limits
L-arginine catabolism (31).
Our studies indicate that BCG exhibits unusual patterns of
regulation of uptake of both GABA and L-arginine. The
GABA utilization genes of B. subtilis, including the permease
gene gabP, are regulated independently by nitrogen starvation
and amino acid availability, and the organisms can use GABA
as the sole nitrogen source (21). Klebsiella aerogenes can use
GABA as both a carbon and nitrogen source, and GABA
genes are induced by GABA in the medium (22). In E. coli,
however, the GABA genes are expressed constitutively at low
levels, and this species is incapable of growing on GABA as a
sole source of either carbon or nitrogen (20). E. coli mutants
with increased expression of the gab regulator (gabC) or the
GABA permease (gabP) can grow on GABA (20). Our study
shows that BCG does not grow with GABA as the sole carbon
and nitrogen source, even at concentrations as high as 50 mM.
Furthermore, incubation of cells with GABA (20 mM) as the
sole carbon and nitrogen source in a minimal salts medium for
2 h or overnight did not increase the uptake of GABA by BCG.
Accumulation of GABA is probably not sufficient to support
the cells in the absence of any other carbon and nitrogen
In B. subtilis, nitrogen starvation causes a 26-fold induction
of expression of the GABA permease, encoded by the gabP
gene. Unlike B. subtilis, the uptake of GABA by BCG was
decreased even under conditions of overnight starvation. BCG
transported GABA most efficiently when the cells were grown
in Middlebrook medium containing appropriate carbon and
nitrogen sources. Therefore, this permease is probably ex-
pressed when cells are in balanced growth in Middlebrook
After incubation in exogenous L-arginine, arginine uptake by
the mutant was dramatically increased to the levels seen in
wild-type cells incubated in L-arginine. These results suggest
that Rv052 may play a role in L-arginine efflux in wild-type
cells: in the ?Rv052 mutant, this efflux activity may be absent,
leading to greatly increased L-arginine uptake after incubation
in exogenous L-arginine. Furthermore, these studies point to
the presence of another L-arginine-responsive permease(s) ex-
pressed in the mutant. One candidate for this permease is a
second rocE homolog discovered in the Sanger database
(Rv2320c). Thus, as in the enterics (11, 53), L-arginine uptake
is carried by more than one permease in BCG. Analysis of the
regulation of expression of the two arginine transporters of M.
tuberculosis and BCG (Rv0522 and Rv2320c) gene is under
It is puzzling that despite the array of L-arginine catabolic
genes, both structural and regulatory, found in the M. tubercu-
losis databases, L-arginine and related amino acids are not
utilized as sole carbon and nitrogen sources by BCG (this
study) or M. tuberculosis (Erdman) (N. D. Connell, unpub-
lished data). In addition, there are no previous reports of
mycobacterial growth inhibition by any amino acids. Lyon et al.
evaluated amino acid utilization by M. tuberculosis, and L-
arginine was among the amino acids tested that were degraded,
as measured by removal of L-arginine, supplied at 5 mM, from
the culture supernatant (33). However, the study did not rely
on the stringent test of utilization of these amino acids as the
sole carbon or nitrogen source, since ammonium and glycerol
were present in the media.
The mechanism by which exogenous L-arginine is growth
inhibitory in BCG is not known. In S. enterica serovar Typhi-
murium, high concentrations of L-arginine (?5 mM) inhibit
the enzyme ornithine carbamoyltransferase (ArgF) (1). High
concentrations of L-arginine (15 mM) repress arginine biosyn-
thetic enzymes 15–20 fold in Streptomyces coelicolor. In BCG,
high concentrations of exogenous L-arginine may repress argi-
nine biosynthesis. L-Arginine accumulation is probably not suf-
ficient for growth but may be high enough to cause a complete
repression of arginine biosynthesis. In many bacterial species,
L-arginine as a corepressor binds to the ArgR repressor to
repress L-arginine biosynthesis (34). Studies of ArgR regula-
tion of L-arginine biosynthesis in BCG and M. tuberculosis are
under way in our laboratory. Alternatively, since the enzymes
involved in L-arginine synthesis are intimately involved in poly-
amine synthesis (24), excess L-arginine may lead to alterations
in polyamine regulation in BCG.
Interestingly, L-arginine biosynthesis and transport have
been shown in a number of pathogenic organisms to be im-
portant in intracellular metabolism. Early IVET studies in S.
enterica serovar Typhimurium, revealed an L-arginine biosyn-
thetic enzyme (carAB) (35). In Listeria monocytogenes, among
the genes expressed preferentially in infected mammalian cells
is arpJ, encoding an L-arginine transporter (30). No L-arginine
biosynthetic mutants have been analyzed in mycobacterial host
cell survival, although an L-arginine tRNA synthetase gene was
VOL. 182, 2000AMINO ACID PERMEASE OF BCG925
specifically induced in M. marinum cells in infected macro-
phages (6). Thus, although shown in different bacterial sys-
tems, genes involved in L-arginine synthesis, transport, and
incorporation into proteins are all increased during intracellu-
lar growth of bacteria. It is well established that L-arginine
transport is stimulated in activated macrophages compared
with resting macrophages, since L-arginine is absolutely re-
quired for the production of nitric oxide in the murine system
(4, 8, 25, 29, 49). AS1 did not show a decrease in survival
compared to wild-type BCG upon infection of J774.1 murine
macrophages. It would be interesting to use multiple mutants
of BCG in which more than one permease has been inactivated
as probes for exploring possible interactions involving L-argi-
nine between the host macrophage and the infecting mycobac-
We thank Marty Pavelka for critical reading of the manuscript. We
also thank the Molecular Resource Facility at the UMD/NJ Medical
School for providing PCR primers and the sequencing facility.
This work was supported in part by Public Health Service Award
2R21AI34436-06A1 to N.D.C., by the Foundation of UMDNJ, and by
the New Jersey Medical School National Tuberculosis Center.
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