scholarly journals Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme.

1987 ◽  
Vol 169 (9) ◽  
pp. 4320-4326 ◽  
Author(s):  
P Lopez ◽  
M Espinosa ◽  
B Greenberg ◽  
S A Lacks
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Sequence ◽  
Gene Encoding ◽  
Dihydropteroate Synthase ◽  
Sulfonamide Resistance

Mechanism of sulfonamide resistance in clinical isolates of Streptococcus pneumoniae.

1997 ◽  
Vol 41 (10) ◽  
pp. 2121-2126 ◽  
Author(s):  
J P Maskell ◽  
A M Sefton ◽  
L M Hall
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Basis ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Gene Exchange ◽  
Gene Encoding ◽  
Dihydropteroate Synthase ◽  
Loss Of Resistance ◽  
Sulfonamide Resistance

The genetic basis of sulfonamide resistance in six clinical isolates of Streptococcus pneumoniae was demonstrated to be 3- or 6-bp duplications within sulA, the chromosomal gene encoding dihydropteroate synthase. The duplications all result in repetition of one or two amino acids in the region from Arg58 to Tyr63, close to but distinct from the sul-d mutation, a duplication previously reported in a resistant laboratory strain (P. Lopez, M. Espinosa, B. Greenberg, and S. A. Lacks, J. Bacteriol. 169:4320-4326, 1987). Six sulfonamide-susceptible clinical isolates lacked such duplications. The role of the duplications in conferring sulfonamide resistance was confirmed by transforming 319- or 322-bp PCR fragments into the chromosome of a susceptible recipient. Two members of a clone of serotype 9V, one susceptible and one resistant to sulfonamide, which are highly related by other criteria, were shown to have sulA sequences that differ in 7.2% of nucleotides in addition to the duplication responsible for resistance. It is postulated that horizontal gene exchange has been involved in the acquisition (or loss) of resistance within this clone. However, five of the six resistant isolates have distinct duplications and other sequence polymorphisms, suggesting that resistance has arisen independently on many occasions.

scholarly journals Novel Expansions of the Gene Encoding Dihydropteroate Synthase in Trimethoprim-Sulfamethoxazole-ResistantStreptococcus pneumoniae

1999 ◽  
Vol 43 (9) ◽  
pp. 2225-2230 ◽  
Author(s):  
Thanugarani Padayachee ◽  
Keith P. Klugman
Keyword(s):  
South African ◽  
Specific Region ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Gene Encoding ◽  
Dihydropteroate Synthase ◽  
Sulfonamide Resistance ◽  
Resistant Strains ◽  
Chromosomal Mutations

ABSTRACT A study of eight sulfonamide-resistant clinical isolates ofStreptococcus pneumoniae revealed chromosomal mutations within the gene encoding dihydropteroate synthase that play a role in conferring resistance to sulfamethoxazole. The presence of thesuld mutation, found previously only in a laboratory mutant, was shown to occur in three of the wild-type clinical isolates. The duplication of Ser61, the other previously defined mutation in the dihydropteroate synthase gene of S. pneumoniae, was observed in only one of the isolates characterized. We report two previously unidentified amino acid alterations, namely, a duplication of Arg58 and Pro59 and an insertion of an arginine residue between Gly60 and Ser61 in trimethoprim-sulfamethoxazole-resistant strains. The significance of these mutations was confirmed by site-directed mutagenesis and by the transformation of a susceptible strain of S. pneumoniae to sulfamethoxazole resistance. Two resistant isolates did not contain any mutations within the gene encoding dihydropteroate synthase. The results presented suggest the independent generation of resistant mutations among South African clinical isolates. It is also proposed that the mechanism of sulfonamide resistance in S. pneumoniae involves the expansion of a specific region within dihydropteroate synthase, which probably forms part of the sulfonamide binding site.

scholarly journals Sulfonamide Resistance in Streptococcus pyogenes Is Associated with Differences in the Amino Acid Sequence of Its Chromosomal Dihydropteroate Synthase

1998 ◽  
Vol 42 (5) ◽  
pp. 1062-1067 ◽  
Author(s):  
Göte Swedberg ◽  
Signe Ringertz ◽  
Ola Sköld
Keyword(s):  
Amino Acid ◽  
Streptococcus Pyogenes ◽  
Gtp Cyclohydrolase ◽  
Gene Encoding ◽  
Additional Indication ◽  
Kinetic Measurements ◽  
Dihydropteroate Synthase ◽  
Development Of Resistance ◽  
Sulfonamide Resistance ◽  
Genes Encoding

ABSTRACT Sulfonamide resistance in recent isolates of Streptococcus pyogenes was found to be associated with alterations of the chromosomally encoded dihydropteroate synthase (DHPS). There were 111 different nucleotides (13.8%) in the genes found in susceptible and resistant isolates, respectively, resulting in 30 amino acid changes (11.3%). These substantial changes suggested the possibility of a foreign origin of the resistance gene, in parallel to what has already been found for sulfonamide resistance in Neisseria meningitidis. The gene encoding DHPS was linked to at least three other genes encoding enzymes of the folate pathway. These genes were in the order GTP cyclohydrolase, dihydropteroate synthase, dihydroneopterin aldolase, and hydroxymethyldihydropterin pyrophosphokinase. The nucleotide differences in genes from resistant and susceptible strains extended from the beginning of the GTP cyclohydrolase gene to the end of the gene encoding DHPS, an additional indication for gene transfer in the development of resistance. Kinetic measurements established different affinities for sulfathiazole for DHPS enzymes isolated from resistant and susceptible strains.

scholarly journals Amino Acid Repetitions in the Dihydropteroate Synthase of Streptococcus pneumoniae Lead to Sulfonamide Resistance with Limited Effects on SubstrateKm

2001 ◽  
Vol 45 (3) ◽  
pp. 805-809 ◽  
Author(s):  
Ylva Haasum ◽  
Katrin Ström ◽  
Rahma Wehelie ◽  
Vicki Luna ◽  
Marilyn C. Roberts ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Washington State ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Directed Mutagenesis ◽  
Aminobenzoic Acid ◽  
Kinetic Measurements ◽  
Dihydropteroate Synthase ◽  
Gene Coding ◽  
Sulfonamide Resistance

ABSTRACT Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniaeisolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile66 and Glu67 in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in Ki for sulfathiazole but changed the Km for p-aminobenzoic acid only 2.5-fold and did not significantly change theKm for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser61 had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.

scholarly journals Phenotypic and Molecular Characterization of Tetracycline- and Erythromycin-Resistant Strains of Streptococcus pneumoniae

2003 ◽  
Vol 47 (7) ◽  
pp. 2236-2241 ◽  
Author(s):  
Maria P. Montanari ◽  
Ileana Cochetti ◽  
Marina Mingoia ◽  
Pietro E. Varaldo
Keyword(s):  
Streptococcus Pneumoniae ◽  
Resistance Gene ◽  
Restriction Analysis ◽  
Tetracycline Resistance ◽  
Erythromycin Resistance ◽  
Gene Encoding ◽  
Conjugative Transposons ◽  
Numerous Type ◽  
The One

ABSTRACT Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and resistant to both tetracycline (MIC, ≥8 μg/ml) and erythromycin (MIC, ≥1 μg/ml), were investigated. Of these strains, 11% were penicillin resistant and 23% were penicillin intermediate. With the use of the erythromycin-clindamycin-rokitamycin triple-disk test, 14 strains were assigned to the constitutive (cMLS) phenotype of macrolide resistance, 44 were assigned to the partially inducible (iMcLS) phenotype, 1 was assigned to the inducible (iMLS) phenotype, and 6 were assigned to the efflux-mediated (M) phenotype. In PCR assays, 64 of the 65 strains were positive for the tetracycline resistance gene tet(M), the exception being the one M isolate susceptible to kanamycin, whereas tet(K), tet(L), and tet(O) were never found. All cMLS, iMcLS, and iMLS isolates had the erythromycin resistance gene erm(B), and all M phenotype isolates had the mef(A) or mef(E) gene. No isolate had the erm(A) gene. The int-Tn gene, encoding the integrase of the Tn916-Tn1545 family of conjugative transposons, was detected in 62 of the 65 test strains. Typing assays showed the strains to be to a great extent unrelated. Of 16 different serotypes detected, the most numerous were 23F (n = 13), 19A (n = 10), 19F (n = 9), 6B (n = 8), and 14 (n = 6). Of 49 different pulsed-field gel electrophoresis types identified, the majority (n = 39) were represented by a single isolate, while the most numerous type included five isolates. By high-resolution restriction analysis of PCR amplicons with four endonucleases, the tet(M) loci from the 64 tet(M)-positive pneumococci were classified into seven distinct restriction types. Overall, a Tn1545-like transposon could reasonably account for tetracycline and erythromycin resistance in the vast majority of the pneumococci of cMLS, iMcLS, and iMLS phenotypes, whereas a Tn916-like transposon could account for tetracycline resistance in most M phenotype strains.

scholarly journals Purification and characterization of an α-l-arabinofuranosidase from Streptomyces lividans 66 and DNA sequence of the gene (abfA)

1994 ◽  
Vol 302 (2) ◽  
pp. 443-449 ◽  
Author(s):  
C Manin ◽  
F Shareek ◽  
R Morosoli ◽  
D Kluepfel
Keyword(s):  
Dna Sequence ◽  
Avena Sativa ◽  
Specific Activity ◽  
Gel Filtration ◽  
Streptomyces Lividans ◽  
Native Protein ◽  
Purification And Characterization ◽  
Gene Encoding ◽  
Sds Page

The gene encoding an alpha-L-arabinofuranosidase (abfA) was homologously cloned in Streptomyces lividans and its DNA sequence was determined. The enzyme was purified from the cytoplasm of the hyperproducing clone S. lividans IAF116. Its M(r) was estimated by gel filtration and found to be approx. 380,000. Since SDS/PAGE indicated a native protein of M(r) 69,000, it can be concluded that the native protein consists of several subunits of that size. The pI value was 4.6. The kinetic constants determined with p-nitrophenyl alpha-L-arabinofuranoside as substrate were a Vmax of 180 units/mg of protein and a Km of 0.6 mM. The specific activity of the purified enzyme on this substrate was 153 units/mg of protein. Optimal enzyme activity was obtained at 60 degrees C and pH 6.0. The enzyme cleaved p-nitrophenyl alpha-L-arabinofuranoside, but had no activity on a variety of other p-nitrophenyl glycosides, except on p-nitrophenyl beta-D-xylopyranoside. The enzyme showed no activity on oat-spelts (Avena sativa) xylan or arabinogalactan, but acted on beet (Beta) arabinan or arabinoxylan. Hydrolysis occurred on arabino-oligoxylosides obtained from oat-splets xylan after digestion with xylanases. Since S. lividans normally does not secrete arabinofuranosidase, this enzyme may play a role in the assimilation of arabinose moieties from arabinose-containing xylo-oligosaccharides generated by beta-xylosidases or xylanases.

scholarly journals New Aspects of the Interplay between Penicillin Binding Proteins, murM, and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Inga Schweizer ◽  
Sebastian Blättner ◽  
Patrick Maurer ◽  
Katharina Peters ◽  
Daniela Vollmer ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Morphological Changes ◽  
Penicillin Resistance ◽  
Penicillin Binding Protein ◽  
Two Component System ◽  
Gene Encoding ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
High Level

ABSTRACT The Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0 ). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murM Hu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murM Hu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2x Hu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2x Hu17 and pbp1a of strain Hu17 (pbp1a Hu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.

scholarly journals Molecular Characterization of the Complete 23F Capsular Polysaccharide Locus of Streptococcus pneumoniae

1998 ◽  
Vol 180 (19) ◽  
pp. 5273-5278 ◽  
Author(s):  
Mario Ramirez ◽  
Alexander Tomasz
Keyword(s):  
Streptococcus Pneumoniae ◽  
Molecular Characterization ◽  
Dna Sequence ◽  
Capsular Polysaccharide ◽  
Open Reading Frames ◽  
The Other ◽  
Reading Frames

ABSTRACT The complete DNA sequence of the capsular locus 23F ofStreptococcus pneumoniae is presented. The 18.6-kbcps23f locus is composed of 18 open reading frames flanked at the 5′ and 3′ ends by the genes dexB andaliA, an arrangement similar to those of some of the other identified cps loci.

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