Adela G. de la Campa

Viridans Group Streptococci Are Donors in Horizontal Transfer of Topoisomerase IV Genes to Streptococcus pneumoniae

ABSTRACT A total of 46 ciprofloxacin-resistant (Cipr) Streptococcus pneumoniae strains were isolated from 1991 to 2001 at the Hospital of Bellvitge. Five of these strains showed unexpectedly high rates of nucleotide variations in the quinolone resistance-determining regions (QRDRs) of their parC, parE, and gyrA genes. The nucleotide sequence of the full-length parC, parE, and gyrA genes of one of these isolates revealed a mosaic structure compatible with an interspecific recombination origin. Southern blot analysis and nucleotide sequence determinations showed the presence of an ant-like gene in the intergenic parE-parC regions of the S. pneumoniae Cipr isolates with high rates of variations in their parE and parC QRDRs. The ant-like gene was absent from typical S. pneumoniae strains, whereas it was present in the intergenic parE-parC regions of the viridans group streptococci (Streptococcus mitis and Streptococcus oralis). These results suggest that the viridans group streptococci are acting as donors in the horizontal transfer of fluoroquinolone resistance genes to S. pneumoniae.

Horizontal Transfer of parC and gyrA in Fluoroquinolone-Resistant Clinical Isolates of Streptococcus pneumoniae

ABSTRACT We have analyzed genetically three clinical isolates (3180, 3870, and 1244) of Streptococcus pneumoniae with high-level ciprofloxacin resistance. Isolates 3180 and 3870 were atypical because of their insolubility in deoxycholate. However, they hybridized specifically with pneumococcal autolysin and pneumolysin gene probes and have typical pneumococcal atpC andatpA gene sequences. Analysis of the complete sequences of the parC and gyrA genes revealed total variations of 8 and 8.7% (isolate 3180) and 7.4 and 3.6% (isolate 3870), respectively, compared to the wild-type strain R6 sequence. The variations observed between the sequences of R6 and isolate 1244 were less than 0.9%. The structure of the gyrA andparC genes from isolates 3180 and 3870 was organized in sequence blocks that show different levels of divergence, suggesting a pattern of recombination. These results are evidence for recombination at the fluoroquinolone target genes in clinical isolates of S. pneumoniae. The genetically related viridans group streptococci could act as a reservoir for fluoroquinolone resistance genes.

Molecular basis of the optochin-sensitive phenotype of pneumococcus : characterization of the genes encoding the F0 complex of the Streptococcus pneumoniae and Streptococcus oralis H+-ATPases

The gene responsible for the optochin‐sensitive (OptS) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F0 complex of an H+‐ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin‐sensitive resistant (OptS/OptR) phenotype. Our results also show that natural as well as laboratory OptR isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F F0 complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (OptS) and M222 (an OptR strain produced by inter‐species recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane‐bound ATPase activity of the S. pneumoniae wild‐type (OptS) strains, and found a 100‐fold difference between OptS and OptR strains, both in growth inhibition and in membrane ATPase resistance.

Fluoroquinolone Resistance in Penicillin-resistant Streptococcus pneumoniae Clones, Spain

Of 75 clones isolated, 1 had ciprofloxacin efflux, and 74 had mutations at the DNA topoisomerase gene.

Initiation of replication of plasmid pLS1. The initiator protein RepB acts on two distant DNA regions.

The broad host range streptococcal plasmid pLS1 encodes the 24.2 kDa protein RepB, which is involved in the initiation of plasmid replication by an asymmetric rolling circle. RepB was overproduced in an Escherichia coli expression system and the protein was purified and characterized. Determination of the amino-terminal sequence of RepB protein showed that translation starts from the first AUG codon, which is preceded by an atypical ribosome-binding site sequence. RepB protein has in vitro-specific endonuclease and topoisomerase-like activities on the plasmid ori(+). Footprinting experiments showed that RepB protein binds to a DNA region that includes three direct repeats of 11 base-pairs. Initiation of replication of pLS1 could start by a RepB-generated specific nick introduced on the plasmid coding strand. However, as a striking difference with other Gram-positive replicons, the nick generated by RepB lies 86 base-pairs upstream from its binding region. To explain the action of RepB at a distance, complex structures of the pLS1 ori(+) are proposed.

Changes in fluoroquinolone-resistant Streptococcus pneumoniae after 7-valent conjugate vaccination, Spain.

Four new genotypes appeared in 2006 after childhood vaccination was begun.

Fitness of Streptococcus pneumoniae Fluoroquinolone-Resistant Strains with Topoisomerase IV Recombinant Genes

ABSTRACT The low prevalence of ciprofloxacin-resistant (Cpr) Streptococcus pneumoniae isolates carrying recombinant topoisomerase IV genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal parE-parC intergenic regions. A study of the transcription of these genes and of the fitness cost for 24 isogenic Cpr strains was performed. Six first-level transformants were obtained either with PCR products containing the parC quinolone resistance-determining regions (QRDRs) of S. pneumoniae Cpr mutants with point mutations or with a PCR product that includes parE-QRDR-ant-parC-QRDR from a CprStreptococcus mitis isolate. The latter yielded two strains, T6 and T11, carrying parC-QRDR and parE-QRDR-ant-parC-QRDR, respectively. These first-level transformants were used as recipients in further transformations with the gyrA-QRDR PCR products to obtain 18 second-level transformants. In addition, strain Tr7 (which contains the GyrA E85K change) was used. Reverse transcription-PCR experiments showed that parE and parC were cotranscribed in R6, T6, and T11; and a single promoter located upstream of parE was identified in R6 by primer extension. The fitness of the transformants was estimated by pairwise competition with R6 in both one-cycle and two-cycle experiments. In the one-cycle experiments, most strains carrying the GyrA E85K change showed a fitness cost; the exception was recombinant T14. In the two-cycle experiments, a fitness cost was observed in most first-level transformants carrying the ParC changes S79F, S79Y, and D83Y and the GyrA E85K change; the exceptions were recombinants T6 and T11. The results suggest that there is no impediment due to a fitness cost for the spread of recombinant CprS. pneumoniae isolates, since some recombinants (T6, T11, and T14) exhibited an ability to compensate for the cost.

The promoter of the operon encoding the F0F1 ATPase of Streptococcus pneumoniae is inducible by pH

The genes encoding the subunits of the FoF1 membrane ATPase of Streptococcus pneumoniae were cloned and sequenced. The eight genes, transcribed to one mRNA, are organized in an operon encoding the c, a, b, delta, alpha, gamma, beta and epsilon subunits of 66, 238, 165, 178, 501, 292, 471 and 139 amino acid residues, respectively, that were expressed in an Escherichia coli system. To investigate the role of the ATPase in the regulation of the intracellular pH, the expression of the operon between pH 5.7 and 7.5 was studied. An increase in both the ATPase activity and the amount of the alpha and beta F1 subunits as shown by Western blot analysis was observed as the pH decreased. These increases were accompanied by an increase in the atp‐specific mRNA, as shown by Northern blot and slot‐blot analysis. Primer extension experiments and transcriptional fusions between the atp promoter and the reporter cat gene demonstrated that this pH‐dependent increase in the mRNA was regulated at the level of initiation of transcription. Transcription of the operon occurs from a promoter with a consensus −35 box (TTGACA) and a −10 box (TACACT) that differs from the consensus (TATAAT). A point mutation at the −10 box of the promoter (change to TGCACT) avoided this increase, suggesting a role for this sequence in the pH‐inducible regulation.

The genome of Streptococcus pneumoniae is organized in topology-reacting gene clusters

The transcriptional response of Streptococcus pneumoniae was examined after exposure to the GyrB-inhibitor novobiocin. Topoisomer distributions of an internal plasmid confirmed DNA relaxation and recovery of the native level of supercoiling at low novobiocin concentrations. This was due to the up-regulation of DNA gyrase and the down-regulation of topoisomerases I and IV. In addition, >13% of the genome exhibited relaxation-dependent transcription. The majority of the responsive genes (>68%) fell into 15 physical clusters (14.6–85.6 kb) that underwent coordinated regulation, independently of operon organization. These genomic clusters correlated with AT content and codon composition, showing the chromosome to be organized into topology-reacting gene clusters that respond to DNA supercoiling. In particular, down-regulated clusters were flanked by 11–40 kb AT-rich zones that might have a putative structural function. This is the first case where genes responding to changes in the level of supercoiling in a coordinated manner were found organized as functional clusters. Such an organization revealed DNA supercoiling as a general feature that controls gene expression superimposed on other kinds of more specific regulatory mechanisms.

Mefloquine and New Related Compounds Target the F0 Complex of the F0F1 H+-ATPase of Streptococcus pneumoniae

ABSTRACT The activities of mefloquine (MFL) and related compounds against previously characterized Streptococcus pneumoniae strains carrying defined amino acid substitutions in the c subunit of the F0F1 H+-ATPase were studied. In addition, a series of MFL-resistant (Mflr) strains were isolated and characterized. A good correlation was observed between inhibition of growth and inhibition of the membrane-associated F0F1 H+-ATPase activity. MFL was about 10-fold more active than optochin and about 200-fold more active than quinine in inhibiting both the growth and the ATPase activities of laboratory pneumococcal strain R6. Mutant strains were inhibited by the different compounds to different degrees, depending on their specific mutations in the c subunit. The resistant strains studied had point mutations that changed amino acid residues in either the c subunit or the a subunit of the F0 complex. Changes in the c subunit were located in one of the two transmembrane α helices: residues M13, G14, G20, M23, and N24 of helix 1 and residues M44, G47, V48, A49, and V57 of helix 2. Changes in the a subunit were also found in either of the transmembrane α helices, helix 5 or 6: residue L186 of helix 5 and residues W206, F209, and S214 of helix 6. These results suggest that the transmembrane helices of the c and a subunits interact and that the mutated residues are important for the structure of the F0 complex and proton translocation.