Laboratory Strain R6 Research Articles

Molecular surveillance of the subtle septum: discovering a new mode of peptidoglycan synthesis in streptococci.

The process of septation requires precise temporal and spatial organization of penicillin binding proteins (PBPs) and associated proteins for the deposition of new cell wall material. In most bacteria, the filamentous protein FtsZ organises PBPs into assemblies at the midcell which then constrict inwards as peptidoglycan is synthesised, eventually closing the septa. Tsui et al. (2014), through the use of fluorescent d-amino acids and high resolution microscopy, report that PBP2x of Streptococcus pneumoniae is directed to a discrete location within the septal aperture during the later stages of cell division. Once at this new site, PBP2x catalyses the de novo synthesis of peptidoglycan, which is imaged by the authors as a central 'spot', distinct from material made by other PBPs at the outer ring. This discovery, which represents a novel mode of cell wall assembly, was made in a directed capsular knockout of strain D39, thereby avoiding potential mechanistic complications in commonly used laboratory strain R6. These findings prompt not only a partial rethink of septum formation in S. pneumoniae, but consideration of the modes of PBP localization and the subtleties that can influence phenotypic study.

The Requirement for Pneumococcal MreC and MreD Is Relieved by Inactivation of the Gene Encoding PBP1a

MreC and MreD, along with the actin homologue MreB, are required to maintain the shape of rod-shaped bacteria. The depletion of MreCD in rod-shaped bacteria leads to the formation of spherical cells and the accumulation of suppressor mutations. Ovococcus bacteria, such as Streptococcus pneumoniae, lack MreB homologues, and the functions of the S. pneumoniae MreCD (MreCD(Spn)) proteins are unknown. mreCD are located upstream from the pcsB cell division gene in most Streptococcus species, but we found that mreCD and pcsB are transcribed independently. Similarly to rod-shaped bacteria, we show that mreCD are essential in the virulent serotype 2 D39 strain of S. pneumoniae, and the depletion of MreCD results in cell rounding and lysis. In contrast, laboratory strain R6 contains suppressors that allow the growth of ΔmreCD mutants, and bypass suppressors accumulate in D39 ΔmreCD mutants. One class of suppressors eliminates the function of class A penicillin binding protein 1a (PBP1a). Unencapsulated Δpbp1a D39 mutants have smaller diameters than their pbp1a(+) parent or Δpbp2a and Δpbp1b mutants, which lack other class A PBPs and do not show the suppression of ΔmreCD mutations. Suppressed ΔmreCD Δpbp1a double mutants form aberrantly shaped cells, some with misplaced peptidoglycan (PG) biosynthesis compared to that of single Δpbp1a mutants. Quantitative Western blotting showed that MreC(Spn) is abundant (≈8,500 dimers per cell), and immunofluorescent microscopy (IFM) located MreCD(Spn) to the equators and septa of dividing cells, similarly to the PBPs and PG pentapeptides indicative of PG synthesis. These combined results are consistent with a model in which MreCD(Spn) direct peripheral PG synthesis and control PBP1a localization or activity.

Influences of Capsule on Cell Shape and Chain Formation of Wild-Type andpcsBMutants of Serotype 2Streptococcus pneumoniae

PcsB is a protein of unknown function that plays a critical role in cell division in Streptococcus pneumoniae and other ovococcus species of Streptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent and pcsB(+) merodiploid strains indicated that pcsB is essential in serotype 2 S. pneumoniae. Quantitative Western blotting of wild-type and epitope-tagged PcsB showed that all PcsB was processed into cell-associated and secreted forms of the same molecular mass and that cell-associated PcsB was moderately abundant and present at approximately 4,900 monomers per cell. Controlled expression and complementation experiments indicated that there was a causative relationship between the severity of defects in cell division and decreasing PcsB amount. These experiments also showed that perturbations of expression of the upstream mreCD genes did not contribute to the cell division defects of pcsB mutants and that mreCD could be deleted. Unexpectedly, capsule influenced the cell shape and chain formation phenotypes of the wild-type D39 strain and mutants underexpressing PcsB or deleted for other genes involved in peptidoglycan biosynthesis, such as dacA. Underexpression of PcsB did not result in changes in the amounts or composition of lactoyl-peptides, which were markedly different in the R6 and D39 strains, and there was no correlation between decreased PcsB amount and sensitivity to penicillin. Finally, microarray analyses indicated that underexpression of PcsB may generate a signal that increases expression of the VicRK regulon, which includes pcsB.

The EssentialtacFGene Is Responsible for the Choline-Dependent Growth Phenotype ofStreptococcus pneumoniae

Streptococcus pneumoniae has an absolute nutritional requirement for choline, and the choline molecules are known to incorporate exclusively into the cell wall and membrane teichoic acids of the bacterium. We describe here the isolation of a mutant of strain R6 in which a single G-->T point mutation in the gene tacF (formerly designated spr1150) is responsible for generating a choline-independent phenotype. The choline-independent phenotype could be transferred to the laboratory strain R6 and to the encapsulated strain D39 by genetic transformation with a PCR product or with a plasmid carrying the mutated tacF gene. The tacF gene product belongs to the protein family of polysaccharide transmembrane transporters (flippases). A model is presented in which TacF is required for the transport of the teichoic acid subunits across the cytoplasmic membrane. According to this model, wild-type TacF has a strict specificity for choline-containing subunits, whereas the TacF present in the choline-independent mutant strain is able to transport both choline-containing and choline-free teichoic acid chains. The proposed transport specificity of parental-type TacF for choline-containing subunits would ensure the loading of the cell wall with teichoic acid chains decorated with choline residues, which appear to be essential for the virulence of this pathogen.

Genome Sequence of Avery's Virulent Serotype 2 Strain D39 ofStreptococcus pneumoniaeand Comparison with That of Unencapsulated Laboratory Strain R6

Streptococcus pneumoniae (pneumococcus) is a leading human respiratory pathogen that causes a variety of serious mucosal and invasive diseases. D39 is an historically important serotype 2 strain that was used in experiments by Avery and coworkers to demonstrate that DNA is the genetic material. Although isolated nearly a century ago, D39 remains extremely virulent in murine infection models and is perhaps the strain used most frequently in current studies of pneumococcal pathogenesis. To date, the complete genome sequences have been reported for only two S. pneumoniae strains: TIGR4, a recent serotype 4 clinical isolate, and laboratory strain R6, an avirulent, unencapsulated derivative of strain D39. We report here the genome sequences and new annotation of two different isolates of strain D39 and the corrected sequence of strain R6. Comparisons of these three related sequences allowed deduction of the likely sequence of the D39 progenitor and mutations that arose in each isolate. Despite its numerous repeated sequences and IS elements, the serotype 2 genome has remained remarkably stable during cultivation, and one of the D39 isolates contains only five relatively minor mutations compared to the deduced D39 progenitor. In contrast, laboratory strain R6 contains 71 single-base-pair changes, six deletions, and four insertions and has lost the cryptic pDP1 plasmid compared to the D39 progenitor strain. Many of these mutations are in or affect the expression of genes that play important roles in regulation, metabolism, and virulence. The nature of the mutations that arose spontaneously in these three strains, the relative global transcription patterns determined by microarray analyses, and the implications of the D39 genome sequences to studies of pneumococcal physiology and pathogenesis are presented and discussed.

A functional dlt operon, encoding proteins required for incorporation of d-alanine in teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the few species within the group of low-G +C gram-positive bacteria reported to contain no d-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for d-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, d-alanine-d-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli 'lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released d-alanine from dltA-proficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G+C gram-positive bacteria, teichoic acids of S. pneumoniae contain d-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides.

Amino acid mutations essential to production of an altered PBP 2X conferring high-level beta-lactam resistance in a clinical isolate of Streptococcus pneumoniae.

Altered penicillin-binding protein 2X (PBP 2X) is a primary beta-lactam antibiotic resistance determinant and is essential to the development of penicillin and cephalosporin resistance in the pneumococcus. We have studied the importance for resistance of 23 amino acid substitutions located in the transpeptidase domain (TD) of PBP 2X from an isolate with high-level resistance, isolate 3191 (penicillin MIC, 16 mug/ml; cefotaxime MIC, 4 microg/ml). Strain R6(2X/2B/1A/mur) (for which the MICs are as described for isolate 3191) was constructed by transforming laboratory strain R6 with all the necessary resistance determinants (altered PBPs 2X, 2B, and 1A and altered MurM) from isolate 3191. Site-directed mutagenesis was used to reverse amino acid substitutions in altered PBP 2X, followed by investigation of the impact of these reversions on resistance levels in R6(2X/2B/1A/mur). Of the 23 substitutions located in the TD of PBP 2X, reversals at six positions decreased the resistance levels in R6(2X/2B/1A/mur). Reversal of the Thr338Pro and Ile371Thr substitutions individually decreased the penicillin and cefotaxime MICs to 2 and 1 microg/ml, respectively, and individually displayed the greatest impact on resistance. To a lesser extent, reversal of the Leu364Phe, Ala369Val, Arg384Gly, and Tyr595Phe substitutions individually also decreased the penicillin and cefotaxime MICs. Reversal at all six positions collectively decreased both the penicillin and the cefotaxime MICs of R6(2X/2B/1A/mur) to 0.06 microg/ml. This study confirms the essential role of altered PBP 2X as a resistance determinant. Our data reveal that, for isolate 3191, the six amino acid substitutions described above are collectively essential to the production of an altered PBP 2X required for high-level resistance to penicillin and cefotaxime.

Identical penicillin-binding domains in penicillin-binding proteins of Streptococcus pneumoniae clinical isolates with different levels of beta-lactam resistance.

We have sequenced the penicillin-binding domains of the complete repertoire of penicillin-binding proteins and MurM from 22 clinical isolates of Streptococcus pneumoniae that span a wide range of beta-lactam resistance levels. Evidence of mosaicism was found in the genes encoding PBP 1a, PBP 2b, PBP 2x, MurM, and, possibly, PBP 2a. Five isolates were found to have identical PBP and MurM sequences, even though the MICs for penicillin G ranged from 0.25 to 2.0 mg/liter. When the sequences encoding PBP 1a, PBP 2b, and PBP 2x from one of these isolates were used to transform laboratory strain R6, the resulting strain had a resistance level higher than that of the less resistant isolates carrying that PBP set but lower than that of the most resistant isolates carrying that PBP set. This result demonstrates that if the R6 strain is arbitrarily defined as the standard genotype, some wild genetic backgrounds can either increase or decrease the PBP-based resistance phenotype.

Genomic Subtraction Followed by Dot Blot Screening of Streptococcus pneumoniae Clinical and Carriage Isolates Identifies Genetic Differences Associated with Strains That Cause Otitis Media

Streptococcus pneumoniae strains are the leading cause of bacterial otitis media, yet little is known about specific bacterial factors important for this disease. We utilized a molecular epidemiological approach involving genomic subtraction of the S. pneumoniae serogroup 19 middle ear strain 5093 against the laboratory strain R6. Resulting subtraction PCR (sPCR) products were used to screen a panel of 93 middle ear, 90 blood, 35 carriage, and 58 cerebrospinal fluid isolates from young children to identify genes found more frequently among middle ear isolates. Probe P41, similar to a hypothetical protein of Brucella melitensis, occurred among 41% of middle ear isolates and was found 2.8 (95% confidence interval [CI], 1.32 to 6.5), 3.3 (95% CI, 1.9 to 5.7), and 1.8 (95% CI, 1.1 to 3.0) times more frequently among middle ear strains than carriage, blood, or meningitis strains, respectively. sPCR fragment H10, similar to an unknown Streptococcus agalactiae protein, was present in 31% of middle ear isolates and occurred 3.6 (95% CI, 1.2 to 11.2), 2.8 (95% CI, 1.5 to 5.4), and 2.6 (95% CI, 1.2 to 5.5) times more often among middle ear isolates than carriage, blood, or meningitis strains, respectively. These studies have identified two genes of potential importance in otitis media virulence. Further studies are warranted to outline the precise role of these genes in otitis media pathogenesis.

Co-infection of primary human adenoid epithelial cells with Influenza A and Streptococcus pneumoniae

Human adenoid epithelial cells (HAEC) from adenoids were used as a model for exploring potential viral/bacterial synergy. Methods: HAEC were grown on a collagen, matrix-coated tissue culture plate until the monolayer became confluent. A differentiated cell population with secretory component, mucin production, and ciliary activity was seen with histochemical staining. Cells were infected with Influenza A/Beijing/H3N2 at an MOI of 0.01 PFU for 24 h. The monolayer was washed and 10 7 Streptococcus pneumoniae added. After a 3-h pneumococcal infection, pneumococci were titered in the supernatant, in the cells after washing (adherent and intracellular bacteria) and in cells treated with antibiotics to destroy adherent bacteria followed by lysis (intracellular bacteria). Results: Over the 3-h infection period, neither pneumococci type 14 (encapsulated) and R6× (unencapsulated) influenced the titer of influenza released nor did influenza affect the bacterial growth. Co-localization of pneumococcal and influenza infection in individual cells was not evident. The capacity varied of pneumococcal strains to adhere and invade the epithelial surface. Encapsulated type 14 from the bloodstream was less invasive than an unencapsulated laboratory strain R6×. Conclusion: The propensity for bacterial superinfection, based on increased adherence or invasion of pneumococci to influenza-infected epithelial cells, could not be explained in the HAEC model.

High-efficiency generation of antibiotic-resistant strains of Streptococcus pneumoniae by PCR and transformation.

We designed a method by which to generate antibiotic-resistant strains of Streptococcus pneumoniae at frequencies 4 orders of magnitude greater than the spontaneous mutation rate. The method is based on the natural ability of this organism to be genetically transformed with PCR products carrying sequences homologous to its chromosome. The genes encoding the targets of ciprofloxacin (parC, encoding the ParC subunit of DNA topoisomerase IV), rifampin (rpoB, encoding the beta subunit of RNA polymerase), and streptomycin (rpsL, encoding the S12 ribosomal protein) from susceptible laboratory strain R6 were amplified by PCR and used to transform the same strain. Resistant mutants were obtained with a frequency of 10(-4) to 10(-5), depending on the fidelity of the DNA polymerase used for PCR amplifications. Ciprofloxacin-resistant mutants, for which the MICs were four-to eightfold higher than that for R6, carried a single mutation of a residue in the quinolone resistance-determining region: S79 (change to A, F, or Y) or D83 (change to N or V). Rifampin-resistant strains, for which the MICs were at least 133-fold higher than that for R6, contained a single mutation within cluster I of rpoB: S482 (change to P), Q486 (change to L), D489 (change to V), or H499 (change to L or Y). Streptomycin-resistant mutants, for which the MICs were at least 64-fold higher than that for R6, carried a mutation at either K56 (change to I, R, or T) or K101 (change to E). PCR products obtained from the mutants were able to transform R6 to resistance with high efficiency (>10(4)). This method could be used to efficiently obtain resistant mutants for any drug whose target is known.

Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyrA, parC, and parE loci.

Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, > or = 8 micrograms/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 micrograms/ml) and trovafloxacin (MICs, 0.5 to 1 microgram/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA, parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.

Genetic studies of cefotaxime resistance in Streptococcus pneumoniae.

In clinical isolates of highly β-lactam-resistant pneumococcal strains, the penicillin-binding proteins (PBP) are usually altered resulting in a decrease in penicillin affinity. PBP gene sequences of alleles conferring resistance are mosaics of pneumococcal and closely related streptococcal sequences. This results from « horizontal transfer » of PBP gene fragments from oral Streptococci toward Streptococcus pneumoniae by DNA induced transformation (1). However, clinical isolates are non isogenic and the strains from which they originated are not known. Therefore it is not possible to correlate resistance levels with specific sequence modification in the PBP genes. In order to bypass such difficulties, a series of independent, spontaneous mutants resistant to high levels of β-lactam were isolated from the laboratory strain R6 by Laible and Hakenbeck (5). A family of strains has been selected for resistance to cefotaxime, a cephalosporin that binds to PBPs other than PBP2b (4). One of the strains, C506, resistant to 1 μg/ml, was used to define the genes that had been mutated during the progressive selection procedure.

Penicillin-binding proteins as resistance determinants in clinical isolates of Streptococcus pneumoniae.

Altered penicillin-binding proteins (PBPs) with reduced affinity for penicillin are encoded by mosaic genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Generally, members of one bacterial clone contain the same mosaic gene. We report here on a serotype 19A clone of penicillin- and multiple-resistant S. pneumoniae prevalent in Hungary, members of which are exceptionally diverse in terms of PBP properties. The pbp2x gene of four 19A isolates was sequenced, and a distinct mosaic structure detected in each case. The pbp2x genes also differed from a homologous gene of a high-level penicillin-resistant S. mitis from Hungary. The contribution of PBPs to resistance development was studied on transformation experiments using the laboratory strain R6 as recipient, and PBP genes from the type 19A isolate Hu11. pbp2x and pbp2b function as primary resistance determinants for different beta-lactams. Secondary transformation with pbp1a increased the resistance level considerably for penicillins and cefotaxime. Chromosomal DNA of a high-level penicillin- and cefotaxime-resistant S. mitis from Hungary also transformed the R6 strain to increased resistance levels, and PBP2x and PBP2b functioned as primary resistance determinants as above. In contrast, high-level cefotaxime resistance appeared to be due to a low affinity PBP2a, indicating that this PBP can also function as a resistance determinant.

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae

Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 μg benzylpenicillin ml −1. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae.

Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 micrograms benzylpenicillin ml-1. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.

Multiple antibiotic resistance in South African strains of Streptococcus pneumoniae: mechanism of resistance to beta-lactam antibiotics.

Multiply antibiotic-resistant strains of Streptococcus pneumoniae appeared in South Africa in 1977. In these organisms resistance to chloramphenicol is caused by an inducible, drug-inactivating enzyme; however, the basis for resistance to other antibiotics is unknown. Pneumococci with increased resistance to beta-lactam antibiotics do not produce beta-lactamases, a finding indicating the presence of intrinsic resistance to these drugs. One approach to the understanding of the mechanism of this resistance was to study the pattern of penicillin-binding proteins (PBPs) in the South African pneumococci. With the use of highly radioactive penicillin to label PBPs in vivo, it was found that the South African pneumococci have a PBP pattern that differs from that of the sensitive laboratory strain R6 in several respects. Differences include (1) a lack of PBPs la and lb; (2) the presence of a new, faster moving protein (lower molecular weight), named PBP lc; (3) an apparent decrease in the affinity of PBP 2a for [3H]penicillin; and (4) a lack of PBP 2b. Taking advantage of the fact that penicillin resistance is a property acquired in a stepwise process, a series of isogenic and progressively more resistant transformants was constructed. DNA from the resistant South African strain 8249 of S. pneumoniae was used as the donor in a series of transformations for which the recipients were either strain R6 or transformants of organisms with lower levels of resistance. In vivo labeling of the PBPs of these transformants revealed a gradual shift from a pattern similar to that of the sensitive strain (in the transformants of lower resistance) to a pattern resembling that of the highly resistant donor strain (in the transformants of higher resistance) as the level of penicillin resistance increased.