described previously [9]. Briefly, UDP-galactose-4-epimerase
is determined by the procedure of Nikaido [26]. For the determination of galactokinase, the method of Wilson and Hogness
is used [11]. Protein is measured by the method of Lowry et al.
[12]. Specific enzyme activities were expressed as units/mg of
protein.
5. Determination of galactose within bacteria: [14C] galactose
(18000 counts per min/mg) is incorporated to a log-phase
culture of S. typhi galE mutants in BHI broth. After every
30-min interval, five 2-L aliquots are harvested. They are centrifuged and washed twice in 0.85% NaCl. The bacteria are
lysed with glass beads (0.17–0.18 mm) in a Sorvall OmniMixer (DuPont, Norwalk, Conn.), and the suspension is centrifuged. The supernatant (SN 1) is recovered. The pellet is
resuspended in distilled water. The cell wall components are
separated from the glass beads by repeated decantation. The
cell wall suspension is then centrifuged at 40,000 Â g for
20 min, and the supernatant (SN 2) is pooled with the supernatant of the first centrifugation. The pellet is suspended in
distilled water and centrifuged at 2000 Â g for 20 min. The
supernatant is then spun down at 40,000 Â g and the pellet is
suspended in water and used for determinations of carbon-14.
The cytoplasmic fraction is removed of protein contamination
by precipitation with 1 N HClO 4 and then is used for determinations of total [14C] galactose. In this fraction, the concentrations of free galactose, galactose-I-phosphate, and UDP
galactose are determined after their separation by ion exchange
chromatography with Dowex 1.
6. Determination of mice virulence: Live bacteria are suspended
in 0.85% NaCl or in 5% mucin and injected intraperitoneally
into female mice of average weight around 18–20 g. Survival
studies are conducted for around seven days. The median lethal
dose (LDso) is estimated by the method of Reed and
Muench [27].
7. Mouse protection test: Female mice weighing 18–20 g are
subjected to immunization by intraperitoneal inoculation of
0.2 ml of a bacterial suspension containing 10
7 live Ty
21 a/ml, or by subcutaneous inoculation of 0.2 ml of a suspension containing 10
8 viable Ty 21a cells. The elimination of
the vaccine strain is determined by serial bacterial counts in liver
and spleen. Four weeks postimmunization, the mice are subjected to secondary challenge of 10
6 viable cells by intraperitoneal injection or by intravenous inoculation of 5 Â 10
7 viable
cells of S. typhi Ty 2. The degree of protection is determined by
the number of mice alive 10 days after challenge.
342
Dipasree Hajra et al.
is determined by the procedure of Nikaido [26]. For the determination of galactokinase, the method of Wilson and Hogness
is used [11]. Protein is measured by the method of Lowry et al.
[12]. Specific enzyme activities were expressed as units/mg of
protein.
5. Determination of galactose within bacteria: [14C] galactose
(18000 counts per min/mg) is incorporated to a log-phase
culture of S. typhi galE mutants in BHI broth. After every
30-min interval, five 2-L aliquots are harvested. They are centrifuged and washed twice in 0.85% NaCl. The bacteria are
lysed with glass beads (0.17–0.18 mm) in a Sorvall OmniMixer (DuPont, Norwalk, Conn.), and the suspension is centrifuged. The supernatant (SN 1) is recovered. The pellet is
resuspended in distilled water. The cell wall components are
separated from the glass beads by repeated decantation. The
cell wall suspension is then centrifuged at 40,000 Â g for
20 min, and the supernatant (SN 2) is pooled with the supernatant of the first centrifugation. The pellet is suspended in
distilled water and centrifuged at 2000 Â g for 20 min. The
supernatant is then spun down at 40,000 Â g and the pellet is
suspended in water and used for determinations of carbon-14.
The cytoplasmic fraction is removed of protein contamination
by precipitation with 1 N HClO 4 and then is used for determinations of total [14C] galactose. In this fraction, the concentrations of free galactose, galactose-I-phosphate, and UDP
galactose are determined after their separation by ion exchange
chromatography with Dowex 1.
6. Determination of mice virulence: Live bacteria are suspended
in 0.85% NaCl or in 5% mucin and injected intraperitoneally
into female mice of average weight around 18–20 g. Survival
studies are conducted for around seven days. The median lethal
dose (LDso) is estimated by the method of Reed and
Muench [27].
7. Mouse protection test: Female mice weighing 18–20 g are
subjected to immunization by intraperitoneal inoculation of
0.2 ml of a bacterial suspension containing 10
7 live Ty
21 a/ml, or by subcutaneous inoculation of 0.2 ml of a suspension containing 10
8 viable Ty 21a cells. The elimination of
the vaccine strain is determined by serial bacterial counts in liver
and spleen. Four weeks postimmunization, the mice are subjected to secondary challenge of 10
6 viable cells by intraperitoneal injection or by intravenous inoculation of 5 Â 10
7 viable
cells of S. typhi Ty 2. The degree of protection is determined by
the number of mice alive 10 days after challenge.
342
Dipasree Hajra et al.