The high level streptomycin resistance gene from<i>Streptococcus pneumoniae</i>is a homologue of the ribosomal protein S12 gene from<i>Escherichia coli</i>

Salles, Catherine; Créancier, Laurent; Claverys, Jean‐Pierre; Méjean, Vincent

doi:10.1093/nar/20.22.6103

Cited by 44 publications

(44 citation statements)

References 7 publications

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“…Chromosomal DNA of the strain under investigation was digested by appropriate restriction endonucleases and ligated under dilute conditions (1 ,ug (29). For direct sequencing of PCR fragments, the reaction was performed with Sequenase as previously described (27). Primer extension analysis.…”

Section: Methodsmentioning

confidence: 99%

The torR gene of Escherichia coli encodes a response regulator protein involved in the expression of the trimethylamine N-oxide reductase genes

et al. 1994

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Section: Methodsmentioning

confidence: 99%

The torR gene of Escherichia coli encodes a response regulator protein involved in the expression of the trimethylamine N-oxide reductase genes

et al. 1994

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“…The upstream and downstream arms of homology flanking cpsE were PCR amplified from TIGR4, MGMV 3105, or MGMV 3526 genomic DNA (gDNA) where appropriate. To aid in making cpsE mutants, a streptomycin resistance (Sm r ) construct, conferred by a point mutation in the rpsL gene (45), was also included during the transformation process. Transformation of S. pneumoniae was done as previously described (46).…”

Section: Methodsmentioning

confidence: 99%

Mutations in Pneumococcal cpsE Generated via In Vitro Serial Passaging Reveal a Potential Mechanism of Reduced Encapsulation Utilized by a Conjunctival Isolate

et al. 2015

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The polysaccharide capsule of Streptococcus pneumoniae is required for nasopharyngeal colonization and for invasive disease in the lungs, blood, and meninges. In contrast, the vast majority of conjunctival isolates are acapsular. The first serotype-specific gene in the capsule operon, cpsE, encodes the initiating glycosyltransferase and is one of the few serotype-specific genes that can tolerate null mutations. This report characterizes a spontaneously arising TIGR4 mutant exhibiting a reduced capsule, caused by a 6-nucleotide duplication in cpsE which results in duplication of Ala and Ile at positions 45 and 46. This strain (AI45dup) possessed more exposed phosphorylcholine and was hypersusceptible to C3 complement deposition compared to the wild type. Accordingly, the mutant was significantly better at forming abiotic biofilms and binding epithelial cells in vitro but was avirulent in a sepsis model. In vitro serial passaging of the wild-type strain failed to reproduce the AI45dup mutation but instead led to a variety of mutants with reduced capsule harboring single nucleotide polymorphisms (SNPs) in cpsE. A single passage in the sepsis model after high-dose inoculation readily yielded revertants of AI45dup with restored wild-type capsule level, but the majority of SNP alleles of cpsE could not revert, suppress, or bypass. Analysis of cpsE in conjunctival isolates revealed a strain with a single missense mutation at amino acid position 377, which was responsible for reduced encapsulation. This study supports the hypothesis that spontaneous, nonreverting mutations in cpsE serve as a form of adaptive mutation by providing a selective advantage to S. pneumoniae in niches where expression of capsule is detrimental. IMPORTANCEWhile the capsule of Streptococcus pneumoniae is required for colonization and invasive disease, most conjunctival isolates are acapsular by virtue of deletion of the entire capsular operon. We show that spontaneous acapsular mutants isolated in vitro harbor mostly nonrevertible single nucleotide polymorphism (SNP) null mutations in cpsE, encoding the initiating glycosyltransferase. From a small collection of acapsular conjunctival isolates, we identified one strain with a complete capsular operon but containing a SNP in cpsE that we show is responsible for the acapsular phenotype. We propose that acapsular conjunctival isolates may arise initially from such nonreverting SNP null mutations in cpsE, which can be followed later by deletion of portions or all of the cps operon. Streptococcus pneumoniae (the pneumococcus) is a Gram-positive bacterium that is frequently found as a commensal organism of the human nasopharynx. However, depending on the site of dissemination, it can also cause various invasive diseases, including pneumonia, otitis media, sepsis, and meningitis (1-4). One critical pneumococcal virulence factor is the capsular polysaccharide (capsule), of which over 90 different serotypes have been characterized. The capsule has many important immune evasion functions, including...

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“…The second screen was carried out with an acapsular mutant from the AC353 strain background (gift of Ram Iyer). An additional Sm-resistant strain into which isolated transposon insertions were transferred was constructed in AC316 by incorporating a point mutation in residue 56 of the RpsL S12 protein (SP0271) (74). In vitro Magellan 5 transposon (also known as pR412 or pEMSPC) transposition reactions were carried out with purified MarC9 transposase essentially as described previously (37).…”

Section: Methodsmentioning

confidence: 99%

Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization

2008

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Asymptomatic colonization of the nasopharynx by Streptococcus pneumoniae precedes pneumococcal disease, yet pneumococcal colonization factors remain poorly understood. Many bacterial infections involve biofilms which protect bacteria from host defenses and antibiotics. To gain insight into the genetics of biofilm formation by S. pneumoniae, we conducted an in vitro screen for biofilm-altered mutants with the serotype 4 clinical isolate TIGR4. In a first screen of 6,000 mariner transposon mutants, we repeatedly isolated biofilm-overproducing acapsular mutants, suggesting that the capsule was antagonistic to biofilm formation. Therefore, we screened 6,500 additional transposon mutants in an S. pneumoniae acapsular background. Following this approach, we isolated 69 insertions in 49 different genes. The collection of mutants includes genes encoding bona fide and putative choline binding proteins, adhesins, synthases of membrane and cell wall components, extracellular and cell wall proteases, efflux pumps, ABC and PTS transporters, and transcriptional regulators, as well as several conserved and novel hypothetical proteins. Interestingly, while four insertions mapped to rrgA, encoding a subunit of a recently described surface pilus, rrgB and rrgC (encoding the other two pilus subunits) mutants had no biofilm defects, implicating the RrgA adhesin but not the pilus structure per se in biofilm formation. To correlate our findings to the process of colonization, we transferred a set of 29 mutations into the wild-type encapsulated strain and then tested the fitness of the mutants in vivo. Strikingly, we found that 23 of these mutants were impaired for nasopharyngeal colonization, thus establishing a link between biofilm formation and colonization.The life cycle of obligate commensal and pathogenic bacteria depends on efficient host colonization and transmission. It is increasingly being recognized that bacteria alternate between planktonic and sessile forms of growth, the latter in the form of surface-adherent biofilms-typically, complex microbial communities sometimes comprised of several species living in symbiotic relationships within a structured extracellular matrix of proteins, polysaccharides, and DNA. To form biofilms on a surface, bacteria rely on multiple genetic systems, including those involving attachment, intercellular interactions (e.g., quorum sensing), chemotaxis, carbon sensing, and stress responses (21,23,67,87). Sessile bacteria differ strikingly physiologically and metabolically from their planktonic counterparts, and the establishment of bacterial biofilms on host tissues is thought to lead to expression of fitness determinants, protect against host defenses, and enhance resistance to antibiotics (29, 67). These observations and the fact that over half of all bacterial infections are believed to involve biofilms make the study of the role of biofilms in host-pathogen interactions an area of major scientific and clinical relevance (29,69).Streptococcus pneumoniae frequently colonizes the human oronas...

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The high level streptomycin resistance gene fromStreptococcus pneumoniaeis a homologue of the ribosomal protein S12 gene fromEscherichia coli

Cited by 44 publications

References 7 publications

The torR gene of Escherichia coli encodes a response regulator protein involved in the expression of the trimethylamine N-oxide reductase genes

The torR gene of Escherichia coli encodes a response regulator protein involved in the expression of the trimethylamine N-oxide reductase genes

Mutations in Pneumococcal cpsE Generated via In Vitro Serial Passaging Reveal a Potential Mechanism of Reduced Encapsulation Utilized by a Conjunctival Isolate

Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization

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