Papers by regine hakenbeck
The human pathogen Streptococcus pneumoniae has been treated for decades with -lactam antibiotic... more The human pathogen Streptococcus pneumoniae has been treated for decades with -lactam antibiotics. Its resistance is now widespread, mediated by the expression of mosaic variants of the target enzymes, the penicillin-binding proteins (PBPs). Understanding the mode of action of -lactams, not only in molecular detail but also in their physiological consequences, will be crucial to improving these drugs and any counterresistances. In this work, we investigate the piperacillin paradox, by which this -lactam selects primarily variants of PBP2b, whereas its most reactive target is PBP2x. These PBPs are both essential monofunctional transpeptidases involved in peptidoglycan assembly. PBP2x participates in septal synthesis, while PBP2b functions in peripheral elongation. The formation of the "lemon"-shaped cells induced by piperacillin treatment is consistent with the inhibition of PBP2x. Following the examination of treated and untreated cells by electron microscopy, the localization of the PBPs by epifluorescence microscopy, and the determination of the inhibition time course of the different PBPs, we propose a model of peptidoglycan assembly that accounts for the piperacillin paradox.
2 Streptococcus oralis, a commensal species of the human oral cavity, belongs to the Mitis group ... more 2 Streptococcus oralis, a commensal species of the human oral cavity, belongs to the Mitis group of streptococci that includes one of the major human pathogens as well, S. pneumoniae. We report here the first complete genome sequence of this species. S. oralis Uo5, a high-level penicillin- and multiple antibiotic-resistant isolate from Hungary, is competent for genetic transformation under laboratory conditions. Comparative and functional genomics of Uo5 will be important in understanding the evolution of pathogenesis among Mitis streptococci, and its potential to engage in interspecies gene transfer. 3 The Mitis phylogenetic group of streptococci consists of members of the commensal flora of the upper respiratory tract of humans (1,2,9). It includes S. pneumoniae, one of the major human pathogens and resident of the nasopharynx, and its closest relatives S. mitis and S. oralis. A detailed in silico comparison of the S. mitis B6 genome with six S. pneumoniae genomes supported the su...
pneumoniae are primary resistance determinants for different classes of beta-lactam antibiotics.
Microorganisms, 2021
Reduced amounts of the essential penicillin-binding protein 2x (PBP2x) were detected in two cefot... more Reduced amounts of the essential penicillin-binding protein 2x (PBP2x) were detected in two cefotaxime-resistant Streptococcus pneumoniae laboratory mutants C405 and C606. These mutants contain two or four mutations in the penicillin-binding domain of PBP2x, respectively. The transcription of the pbp2x gene was not affected in both mutants; thus, the reduced PBP2x amounts were likely due to post-transcriptional regulation. The mutants carry a mutation in the histidine protein kinase gene ciaH, resulting in enhanced gene expression mediated by the cognate response regulator CiaR. Deletion of htrA, encoding a serine protease regulated by CiaR, or inactivation of HtrA proteolytic activity showed that HtrA is indeed responsible for PBP2x degradation in both mutants, and that this affects β-lactam resistance. Depletion of the PBP2xC405 in different genetic backgrounds confirmed that HtrA degrades PBP2xC405. A GFP-PBP2xC405 fusion protein still localized at the septum in the absence of Ht...
Journal of Bacteriology, 1977
Inhibition of peptidoglycan synthesis causes an immediate and massive secretion of both newly syn... more Inhibition of peptidoglycan synthesis causes an immediate and massive secretion of both newly synthesized and "old" lipids from several species of bacteria, including streptococci, Staphylococcus epidermidis, and Bacillus subtilis. Lipid secretion occurs in the absence of detectable bacterial lysis. This novel phenomenon was examined in more detail in three strains of streptococci: S. sanguis (group H), S. pyogenes (group A), And S. pneumoniae. The secretion of lipids is specifically induced by inhibitors of peptidoglycan synthesis; it is not caused by inhibitors of protein, ribonucleic acid, or deoxyribonucleic acid synthesis. The occurrence appears to be reversible since penicillin-induced secretion comes to a halt upon the timely addition of penicillinase, correlating with resumption of culture growth. All cellular lipids are secreted in essentially the same proportions as those found in the drug treated bacteria. It is suggested that continued peptidoglycan synthesis m...
Journal of Bacteriology, 1998
Penicillin-resistant isolates of Streptococcus pneumoniae generally contain mosaic genes encoding... more Penicillin-resistant isolates of Streptococcus pneumoniae generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in β-lactam-resistant transformants of S. pneumoniae obtained with chromosomal donor DNA from a Streptococcus mitis isolate. Different lineages of β-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzylpenicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 μg/ml, respectively, close to the MICs for the S. mitis donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qual...
Journal of Bacteriology, 1999
ABSTRACTOne group of penicillin target enzymes, the class A high-molecular-weight penicillin-bind... more ABSTRACTOne group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding–transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the threeStreptococcus pneumoniaeclass A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disruptedpbp2agene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors inS. pneumoniae.
Antimicrobial Agents and Chemotherapy, 1997
Cefotaxime resistance in laboratory mutant C604 of Streptococcus pneumoniae, for which the MIC is... more Cefotaxime resistance in laboratory mutant C604 of Streptococcus pneumoniae, for which the MIC is 1.5 microg/ml, is independent of alterations in high-molecular-mass penicillin-binding protein (PBP) 1a. Instead, a point mutation in PBP 3, the D,D-carboxypeptidase of this organism, caused a reduced affinity for penicillin and contributed to the decreased susceptibility. The mutation Thr-242 to Ile was located directly adjacent to the triad Lys-239-Thr-Gly, a position known to be important for beta-lactam interaction with high-molecular-mass PBPs and beta-lactamases. This mutation was absent in the PBP 3's of four genetically distinct clinical isolates resistant to high levels of penicillin. None of the pbp3 genes had a mosaic structure, but in three cases there was evidence for a site-specific recombination event within a BOX element immediately downstream of pbp3.
Antimicrobial Agents and Chemotherapy, 1996
High-level resistance to beta-lactam antibiotics in Streptococcus pneumoniae is mediated by succe... more High-level resistance to beta-lactam antibiotics in Streptococcus pneumoniae is mediated by successive alterations in essential penicillin-binding proteins (PBPs). In the present work, single amino acid changes in S. pneumoniae PBP 2x and PBP 2b that result in reduced affinity for the antibiotic and that confer first-level beta-lactam resistance are defined. Point mutations in the PBP genes were generated by PCR-derived mutagenesis. Those conferring maximal resistance to either cefotaxime (pbp2x) or piperacillin (pbp2b) were obtained after transformation of the susceptible laboratory strain R6 with the PCR-amplified PBP genes and selection on agar with various concentrations of the antibiotic. In the case of PBP 2x, transformants for which the cefotaxime MIC was 0.16 microgram/ml contained the substitution of a Thr for an Ala at position 550 (Thr550-->Ala), close to the PBP homology box Lys547SerGly, a mutation frequently observed in laboratory mutants and in a high-level cefotax...
Antimicrobial agents and chemotherapy, 2017
Penicillin-resistant strains are found at high rates in Romania and Iran. The mosaic structure of... more Penicillin-resistant strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for β-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain-1 clone, which is widespread among international penicillin-resistant clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documen...
mSphere, 2017
Streptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can res... more Streptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor.
Journal of Bacteriology, 1992
The nucleotide sequence of a 3,378-bp DNA fragment of Streptococcus pneumoniae that included the ... more The nucleotide sequence of a 3,378-bp DNA fragment of Streptococcus pneumoniae that included the structural gene for penicillin-binding protein (PBP) 1a (ponA), which encodes 719 amino acids, was determined. Homologous DNA fragments from an S. oralis strain were amplified with ponA-specific oligonucleotides. The 2,524-bp S. oralis sequence contained the coding region for the first 636 amino acids of a PBP. The coding sequence differed by 437 nucleotides (27%) and one additional triplet, resulting in 87 amino acid substitutions (14%), from S. pneumoniae PBP 1a. Both PBPs are highly homologous to bifunctional high-M(r) Escherichia coli PBPs 1a and 1b.
Antimicrobial agents and chemotherapy, 2017
The identification of commensal streptococci species is an everlasting problem due to their abili... more The identification of commensal streptococci species is an everlasting problem due to their ability to genetically transform. A new challenge in this respect is the recent description of Streptococcus pseudopneumoniae as a new species, which was distinguished from closely related pathogenic S. pneumoniae and commensal S. mitis by a variety of physiological and molecular biological tests. Forty-one atypical S. pneumoniae isolates have been collected at the German National Reference Center for Streptococci (GNRCS). Multilocus sequence typing (MLST) confirmed 35 isolates as the species S. pseudopneumoniae A comparison with the pbp2x sequences from 120 commensal streptococci isolated from different continents revealed that pbp2x is distinct among penicillin-susceptible S. pseudopneumoniae isolates. Four penicillin-binding protein x (PBPx) alleles of penicillin-sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pseudopneumoniae, S. pneumoniae, and S. mitis...
Journal of Bacteriology, 1992
The muropeptide composition of a Streptococcus pneumoniae mutant in which the DD-carboxypeptidase... more The muropeptide composition of a Streptococcus pneumoniae mutant in which the DD-carboxypeptidase (penicillin-binding protein 3) gene was interrupted by plasmid insertion close to the 3' end of the gene was examined. Extensive compositional changes were observed: the linear pentapeptide, a minor component of the parental cells, became the most abundant monomeric peptide in the mutant wall, while the proportion of tripeptides that represent the main monomers in the parental cells was greatly reduced. The amount of the major dimer of parental cells, the directly cross-linked tri-tetrapeptide, was also reduced by a factor of 4. It was partially replaced by a novel dimer: the cross-linked product of a linear pentapeptide and a pentapeptide carrying a serylalanine dipeptide substituent on the epsilon-NH2 group of its lysine residue. This dimer together with two other dimeric peptides, each containing the serylalanine cross bridge, became the quantitatively major components of the mut...
Journal of Bacteriology, 1991
Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-w... more Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance ...
Journal of Bacteriology, 1997
Piperacillin resistance in Streptococcus pneumoniae was mediated by mutations in a novel gene, cp... more Piperacillin resistance in Streptococcus pneumoniae was mediated by mutations in a novel gene, cpoA, that also confer transformation deficiency and a decrease in penicillin-binding protein la. cpoA is part of an operon located downstream of the primary sigma factor of S. pneumoniae. The deduced protein, CpoA, and the peptide encoded by the adjacent 3' open reading frame contained domains homologous to glycosyltransferases of procaryotes and eucaryotes that act on membrane-associated substrates, such as enzymes functioning in lipopolysaccharide core biosynthesis of gram-negative bacteria, RodD of Bacillus subtilis, which is involved in teichoic acid biosynthesis, and the human PIG-A protein, which is required for early steps of glycosylphosphatidylinositol anchor biosynthesis. This suggests that the cpo operon has a similar function related to cell surface components.
Journal of Bacteriology, 1997
To map the incidence of natural competence in the genus Streptococcus, we used PCR to screen a nu... more To map the incidence of natural competence in the genus Streptococcus, we used PCR to screen a number of streptococcal strains for the presence of the recently identified competence regulation operon, containing the comC, -D, and -E genes. This approach established that the operon is present in strains belonging to the S. mitis and S. anginosus groups, but it was not detected in the other strains examined. Competence is induced in S. pneumoniae and S. gordonii by strain-specific peptide pheromones, competence-stimulating peptides (CSPs). With its unique primary structure, each CSP represents a separate pheromone type (pherotype), which is recognized by the signalling domain of the downstream histidine kinase, ComD. Thus, all bacteria induced to competence by a particular CSP belong to the same pherotype. In this study, we identified a number of new pherotypes by sequencing the genes encoding the CSP and its receptor from different streptococcal species. We found that in several case...
Journal of Bacteriology, 1997
A gene, mgt, encoding a protein homologous to the N-terminal module of class A high-molecular-mas... more A gene, mgt, encoding a protein homologous to the N-terminal module of class A high-molecular-mass penicillin-binding proteins was identified in Ralstonia eutropha. By using specific antibodies, the corresponding Mgt protein was detected in association with the membrane, confirming that the N-terminal hydrophobic segment functioned as a membrane anchor. A derivative in which the hydrophobic sequence was deleted was overexpressed as a maltose-binding fusion protein in Escherichia coli. Cleavage of the product resulted in substantial amounts of soluble Mgt derivative, indicating that folding occurs independently on other proteins or on homologous domains of penicillin-binding proteins.
mSphere
Viridans streptococci were obtained from primates (great apes, rhesus monkeys, and ring-tailed le... more Viridans streptococci were obtained from primates (great apes, rhesus monkeys, and ring-tailed lemurs) held in captivity, as well as from free-living animals (chimpanzees and lemurs) for whom contact with humans is highly restricted. Isolates represented a variety of viridans streptococci, including unknown species. Streptococcus oralis was frequently isolated from samples from great apes. Genotypic methods revealed that most of the strains clustered on separate lineages outside the main cluster of human S. oralis strains. This suggests that S. oralis is part of the commensal flora in higher primates and evolved prior to humans. Many genes described as virulence factors in Streptococcus pneumoniae were present also in other viridans streptococcal genomes. Unlike in S. pneumoniae, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) gene clusters were common among viridans streptococci, and many S. oralis strains were type PI-2 (pilus isle...
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Papers by regine hakenbeck