Comparison of epidemic and endemic group G streptococci by restriction enzyme analysis

Martin, Nancy J.; Kaplan, Edward L.; Gerber, Michael A.; Menegus, Marilyn A.; Randolph, Martin F.; Bell, Karen; Cleary, P. Patrick

doi:10.1128/jcm.28.9.1881-1886.1990

Cited by 66 publications

(21 citation statements)

References 24 publications

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“…Standard recombinant DNA techniques were employed for nucleic acid preparation and analysis. Genomic streptococcal DNA was isolated as described previously [8]. Plasmid DNA was isolated and puri¢ed using the Qiaprep Spin Miniprep kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.…”

Section: Dna Techniquesmentioning

confidence: 99%

The cyl genes of Streptococcus agalactiae are involved in the production of pigment

Spellerberg

2000

FEMS Microbiology Letters

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The cyl genes of Streptococcus agalactiae are required for the production of hemolysin. Based on the observation that nonhemolytic S. agalactiae mutants do not produce pigment, a close genetic linkage between hemolysin and pigment has been postulated. To investigate this genetic linkage and to identify genes involved in the production of the S. agalactiae pigment, we screened mutant libraries for nonpigmented clones. Four distinct mutants were isolated with a nonpigmented and nonhemolytic phenotype. The mutations had occurred either in known cyl genes or in two open reading frames located immediately downstream. These novel genes are cotranscribed with the cyl gene cluster and were designated cylF and cylI. Our data indicate that identical genes participate in the production of S. agalactiae hemolysin and pigment. ß

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Section: Dna Techniquesmentioning

confidence: 99%

The cyl genes of Streptococcus agalactiae are involved in the production of pigment

Spellerberg

2000

FEMS Microbiology Letters

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“…Chromosomal GAS DNA was prepared according to the method of Martin et al (1990). Plasmid DNA preparations, genetic manipulations and other conventional DNA techniques were performed as described by Ausubel et al (1990).…”

Section: Dna Techniquesmentioning

confidence: 99%

The group A streptococcal dipeptide permease (Dpp) is involved in the uptake of essential amino acids and affects the expression of cysteine protease

Podbielski¹,

Leonard²

1998

Molecular Microbiology

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SummaryThe majority of characterized bacterial dipeptide permeases (Dpp) are membrane-associated complexes of five proteins belonging to the ABC-transporter family. They have been found to be involved in the uptake of essential amino acids, haem production, chemotaxis and sporulation. A 5.8 kb genomic DNA fragment of the serotype M49 group A streptococcal (GAS) strain CS101 was sequenced and found to contain five putative GAS Dpp genes (dppA to dppE ). Deduced amino acid sequences exhibited 17-54% similarity to corresponding ABC-transporter sequences. The operon organization of the five genes was confirmed by transcriptional analysis, and a shorter, more abundant, dppA-only transcript was detected similar to that found in the GAS oligopeptide permease (Opp) system. Insertional inactivation was used to create serotype M2 and M49 strains that did not express the dppD and dppE ATPase genes or nearly the entire operon. In feeding experiments with di-to hexapeptides, the wild-type strain grew with each peptide tested. The dpp mutants were unable to grow on dipeptides, whereas hexapeptides did not sustain the growth of opp mutants. Expression of the dpp operon was induced approximately fourfold in late exponential growth phase. In addition, a striking increase in the dppA to dppA-E ratio from 5:1 to more than 20:1 occurred during late exponential growth phase in complex medium. Growth in chemically defined medium (CDM) supplemented with various dipeptides specifically induced the expression of dpp and reduced both the dppA to dppA-E and oppA to oppA-F mRNA ratios. Expression of the virulence factor SpeB (major cysteine protease) was reduced eightfold in dpp mutants, whereas dpp expression was decreased about fourfold in a Mga virulence regulator mutant. Taken together, these data indicate a correlation between levels of intracellular essential amino acids and the regulation of virulence factor expression.

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“…Chromosomal GAS DNA was prepared by the method of Martin et al (1990), and plasmid DNA from E. coli recombinants was prepared according to Zhou et al (1990). Conventional techniques for DNA manipulations such as restriction enzyme digests, ligation, transformation of E. coli host cells, colony blot hybridization, 1% agarose gel electrophoresis, and Southern blotting by vacuum transfer were performed as outlined by Ausubel et al (1990).…”

Section: Dna Techniquesmentioning

confidence: 99%

M‐related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes

Podbielski¹,

Schnitzler²,

Beyhs³

et al. 1996

Molecular Microbiology

109

132

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The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsonophagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co-incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.

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Comparison of epidemic and endemic group G streptococci by restriction enzyme analysis

Cited by 66 publications

References 24 publications

The cyl genes of Streptococcus agalactiae are involved in the production of pigment

The cyl genes of Streptococcus agalactiae are involved in the production of pigment

The group A streptococcal dipeptide permease (Dpp) is involved in the uptake of essential amino acids and affects the expression of cysteine protease

M‐related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes

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