strain cwg304  (ATCC)

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: (A) Structure of the LPS core oligosaccharide from S. enterica serovar Typhimurium (20). The inner core heptose region is highlighted by shading. Genetic determinants for the modification of the inner core heptose region (conserved between E. coli and S. enterica) are shown, and they must function in the following sequence: waaP, waaQ, and then waaY (43). Abbreviations: Hep, l-glycero-d-manno-heptose; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; P, phosphate; PS, polysaccharide. ∗, the PEtN substitution is nonstoichiometric but is reportedly present in larger amounts in PM-resistant mutants of S. enterica (16). (B) Predicted structure of the LPS core oligosaccharide from CWG304, based on the function of WaaP in E. coli F470 and its effect on WaaQ and WaaY activities (43).

Article Snippet: To determine whether such was the case for our defined mutant strain (CWG304), we examined the CWG304 and parent (ATCC 14028) LPSs by SDS-PAGE (Fig. ).

Techniques: Modification, Sequencing

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: Silver-stained SDS-polyacrylamide gels showing the LPS profiles of strains ATCC 14028 (lane 1), CWG304 (lane 2), and CWG304 complemented with the waaP open reading frame from E. coli F470 (lane 3). Samples were run on a 10 to 20% Tricine SDS-polyacrylamide gel (A) and on a standard SDS–12% polyacrylamide gel (B). The gel system in panel A gives better resolution of low-molecular-weight LPS (i.e., LPS lacking O antigen), while the standard gel system shows that the modality of O-antigen expression is unaffected in the mutant strain. The migration of Ra-LPS (lipid A and complete core) in each gel system is indicated by an arrow. Note that Ra-LPS comigrates with LPS molecules with one O-antigen repeat in the gel system in panel B, so the amount of free (uncapped) lipid A-core is misleading. The extent of capping is clear in panel A.

Article Snippet: To determine whether such was the case for our defined mutant strain (CWG304), we examined the CWG304 and parent (ATCC 14028) LPSs by SDS-PAGE (Fig. ).

Techniques: Staining, Molecular Weight, Expressing, Mutagenesis, Migration

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: 31P NMR spectra of the core oligosaccharides from ATCC 14028 (A) and CWG304 (B). The signal at 5 ppm is indicative of a phosphomonoester (P on either HepI or HepII), and the two peaks near −10 ppm are characteristic of a diphosphodiester (PPEtN on HepI) (16).

Article Snippet: To determine whether such was the case for our defined mutant strain (CWG304), we examined the CWG304 and parent (ATCC 14028) LPSs by SDS-PAGE (Fig. ).

Techniques:

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: Methylation linkage analysis of the core oligosaccharide heptose regions from the LPSs of ATCC 14028 and CWG304

Article Snippet: To determine whether such was the case for our defined mutant strain (CWG304), we examined the CWG304 and parent (ATCC 14028) LPSs by SDS-PAGE (Fig. ).

Techniques: Methylation

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: MIC testing for susceptibility to novobiocin, SDS, and PM

Article Snippet: To determine whether such was the case for our defined mutant strain (CWG304), we examined the CWG304 and parent (ATCC 14028) LPSs by SDS-PAGE (Fig. ).

Techniques:

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: Characterization of structural modifications of S. enterica lipid A by negative-ion MALDI-TOF mass spectrometry. All values given are average masses rounded to the nearest whole number for singly charged, deprotonated molecules [M − H]−. (A) ATCC 14028 (parent) lipid A, with the major signal representing the hexa-acylated form (at m/z 1797). The hepta-acylated form containing palmitate (at m/z 2036) is indicated. (B) CWG304 (waaP::aacC1) lipid A, showing ions at m/z 1797 and 2036 as described above. (C) JSG435 (pmrA505) lipid A, showing the hexa- and hepta-acylated forms (as described above), as well as modification by the addition of aminoarabinose (m/z 1928 and 2167, respectively) or by the addition of a hydroxyl group (m/z 1814 and 2052, respectively). (D) JSG778 (pmrA505 waaP::aacC1) lipid A, showing ions at m/z 1797, 1814, 1928, 2036, 2052, and 2167 as described above.

Article Snippet: To determine whether such was the case for our defined mutant strain (CWG304), we examined the CWG304 and parent (ATCC 14028) LPSs by SDS-PAGE (Fig. ).

Techniques: Mass Spectrometry, Modification

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: Average numbers of CFU in the spleens and livers of A/J mice challenged intravenously with the indicated doses of ATCC 14028 or CWG304 a

Article Snippet: To determine whether such was the case for our defined mutant strain (CWG304), we examined the CWG304 and parent (ATCC 14028) LPSs by SDS-PAGE (Fig. ).

Techniques:

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: (A) Structure of the LPS core oligosaccharide from S. enterica serovar Typhimurium (20). The inner core heptose region is highlighted by shading. Genetic determinants for the modification of the inner core heptose region (conserved between E. coli and S. enterica) are shown, and they must function in the following sequence: waaP, waaQ, and then waaY (43). Abbreviations: Hep, l-glycero-d-manno-heptose; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; P, phosphate; PS, polysaccharide. ∗, the PEtN substitution is nonstoichiometric but is reportedly present in larger amounts in PM-resistant mutants of S. enterica (16). (B) Predicted structure of the LPS core oligosaccharide from CWG304, based on the function of WaaP in E. coli F470 and its effect on WaaQ and WaaY activities (43).

Article Snippet: However, CWG304 bacteria were virtually cleared by day 21 and could be detected only in very small numbers in the spleen at the highest dose administered. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Dose (CFU) Avg CFU b per: Spleen Liver ATCC 14028 CWG304 ATCC 14028 CWG304 10 4 3,468 ± 3,352 c 9 ± 5 142 ± 46 0 10 3 56 ± 13 0 32 ± 17 0 10 2 58 ± 9 0 31 ± 3 0 Open in a separate window a Surviving mice were sacrificed at 21 days postinfection. b Results are averages and standard deviations from five mice, except as indicated. c Only two of five mice survived until day 21 at this dose.

Techniques: Modification, Sequencing

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: Silver-stained SDS-polyacrylamide gels showing the LPS profiles of strains ATCC 14028 (lane 1), CWG304 (lane 2), and CWG304 complemented with the waaP open reading frame from E. coli F470 (lane 3). Samples were run on a 10 to 20% Tricine SDS-polyacrylamide gel (A) and on a standard SDS–12% polyacrylamide gel (B). The gel system in panel A gives better resolution of low-molecular-weight LPS (i.e., LPS lacking O antigen), while the standard gel system shows that the modality of O-antigen expression is unaffected in the mutant strain. The migration of Ra-LPS (lipid A and complete core) in each gel system is indicated by an arrow. Note that Ra-LPS comigrates with LPS molecules with one O-antigen repeat in the gel system in panel B, so the amount of free (uncapped) lipid A-core is misleading. The extent of capping is clear in panel A.

Article Snippet: However, CWG304 bacteria were virtually cleared by day 21 and could be detected only in very small numbers in the spleen at the highest dose administered. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Dose (CFU) Avg CFU b per: Spleen Liver ATCC 14028 CWG304 ATCC 14028 CWG304 10 4 3,468 ± 3,352 c 9 ± 5 142 ± 46 0 10 3 56 ± 13 0 32 ± 17 0 10 2 58 ± 9 0 31 ± 3 0 Open in a separate window a Surviving mice were sacrificed at 21 days postinfection. b Results are averages and standard deviations from five mice, except as indicated. c Only two of five mice survived until day 21 at this dose.

Techniques: Staining, Molecular Weight, Expressing, Mutagenesis, Migration

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: 31P NMR spectra of the core oligosaccharides from ATCC 14028 (A) and CWG304 (B). The signal at 5 ppm is indicative of a phosphomonoester (P on either HepI or HepII), and the two peaks near −10 ppm are characteristic of a diphosphodiester (PPEtN on HepI) (16).

Article Snippet: However, CWG304 bacteria were virtually cleared by day 21 and could be detected only in very small numbers in the spleen at the highest dose administered. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Dose (CFU) Avg CFU b per: Spleen Liver ATCC 14028 CWG304 ATCC 14028 CWG304 10 4 3,468 ± 3,352 c 9 ± 5 142 ± 46 0 10 3 56 ± 13 0 32 ± 17 0 10 2 58 ± 9 0 31 ± 3 0 Open in a separate window a Surviving mice were sacrificed at 21 days postinfection. b Results are averages and standard deviations from five mice, except as indicated. c Only two of five mice survived until day 21 at this dose.

Techniques:

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: Methylation linkage analysis of the core oligosaccharide heptose regions from the LPSs of ATCC 14028 and CWG304

Article Snippet: However, CWG304 bacteria were virtually cleared by day 21 and could be detected only in very small numbers in the spleen at the highest dose administered. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Dose (CFU) Avg CFU b per: Spleen Liver ATCC 14028 CWG304 ATCC 14028 CWG304 10 4 3,468 ± 3,352 c 9 ± 5 142 ± 46 0 10 3 56 ± 13 0 32 ± 17 0 10 2 58 ± 9 0 31 ± 3 0 Open in a separate window a Surviving mice were sacrificed at 21 days postinfection. b Results are averages and standard deviations from five mice, except as indicated. c Only two of five mice survived until day 21 at this dose.

Techniques: Methylation

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: MIC testing for susceptibility to novobiocin, SDS, and PM

Article Snippet: However, CWG304 bacteria were virtually cleared by day 21 and could be detected only in very small numbers in the spleen at the highest dose administered. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Dose (CFU) Avg CFU b per: Spleen Liver ATCC 14028 CWG304 ATCC 14028 CWG304 10 4 3,468 ± 3,352 c 9 ± 5 142 ± 46 0 10 3 56 ± 13 0 32 ± 17 0 10 2 58 ± 9 0 31 ± 3 0 Open in a separate window a Surviving mice were sacrificed at 21 days postinfection. b Results are averages and standard deviations from five mice, except as indicated. c Only two of five mice survived until day 21 at this dose.

Techniques:

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: Characterization of structural modifications of S. enterica lipid A by negative-ion MALDI-TOF mass spectrometry. All values given are average masses rounded to the nearest whole number for singly charged, deprotonated molecules [M − H]−. (A) ATCC 14028 (parent) lipid A, with the major signal representing the hexa-acylated form (at m/z 1797). The hepta-acylated form containing palmitate (at m/z 2036) is indicated. (B) CWG304 (waaP::aacC1) lipid A, showing ions at m/z 1797 and 2036 as described above. (C) JSG435 (pmrA505) lipid A, showing the hexa- and hepta-acylated forms (as described above), as well as modification by the addition of aminoarabinose (m/z 1928 and 2167, respectively) or by the addition of a hydroxyl group (m/z 1814 and 2052, respectively). (D) JSG778 (pmrA505 waaP::aacC1) lipid A, showing ions at m/z 1797, 1814, 1928, 2036, 2052, and 2167 as described above.

Article Snippet: However, CWG304 bacteria were virtually cleared by day 21 and could be detected only in very small numbers in the spleen at the highest dose administered. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Dose (CFU) Avg CFU b per: Spleen Liver ATCC 14028 CWG304 ATCC 14028 CWG304 10 4 3,468 ± 3,352 c 9 ± 5 142 ± 46 0 10 3 56 ± 13 0 32 ± 17 0 10 2 58 ± 9 0 31 ± 3 0 Open in a separate window a Surviving mice were sacrificed at 21 days postinfection. b Results are averages and standard deviations from five mice, except as indicated. c Only two of five mice survived until day 21 at this dose.

Techniques: Mass Spectrometry, Modification

Journal:

Article Title: Salmonella enterica Serovar Typhimurium waaP Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo

doi:

Figure Lengend Snippet: Average numbers of CFU in the spleens and livers of A/J mice challenged intravenously with the indicated doses of ATCC 14028 or CWG304 a

Article Snippet: However, CWG304 bacteria were virtually cleared by day 21 and could be detected only in very small numbers in the spleen at the highest dose administered. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Dose (CFU) Avg CFU b per: Spleen Liver ATCC 14028 CWG304 ATCC 14028 CWG304 10 4 3,468 ± 3,352 c 9 ± 5 142 ± 46 0 10 3 56 ± 13 0 32 ± 17 0 10 2 58 ± 9 0 31 ± 3 0 Open in a separate window a Surviving mice were sacrificed at 21 days postinfection. b Results are averages and standard deviations from five mice, except as indicated. c Only two of five mice survived until day 21 at this dose.

Techniques: