Molecular Bulkiness of a Single Amino Acid in the F1 α-Subunit Determines the Robustness of Cyanobacterial ATP Synthase.

Cyanobacteria are promising photosynthetic organisms owing to their ease of genetic manipulation. Among them, Synechococcus elongatus UTEX 2973 exhibits faster growth, higher biomass production efficiency and more robust stress tolerance compared with S. elongatus PCC 7942. This is due to specific genetic differences, including four single-nucleotide polymorphisms (SNPs) in three genes. One of these SNPs alters an amino acid at position 252 of the FoF1 ATP synthase α-subunit from Tyr to Cys (αY252C) in S. elongatus 7942. This change has been shown to significantly affect growth rate and stress tolerance, specifically in S. elongatus. Furthermore, experimental substitutions with several other amino acids have been shown to alter the ATP synthesis rate in the cell. In the present study, we introduced identical amino acid substitutions into Synechocystis sp. PCC 6803 at position 252 to elucidate the amino acid's significance and generality across cyanobacteria. We investigated the resulting impact on growth, intracellular enzyme complex levels, intracellular ATP levels and enzyme activity. The results showed that the αY252C substitution decreased growth rate and high-light tolerance. This indicates that a specific bulkiness of this amino acid's side chain is important for maintaining cell growth. Additionally, a remarkable decrease in the membrane-bound enzyme complex level was observed. However, the αY252C substitution did not affect enzyme activity or intracellular ATP levels. Although the mechanism of growth suppression remains unknown, the amino acid at position 252 is expected to play an important role in enzyme complex formation.

Two specific domains of the γ subunit of chloroplast FoF1 provide redox regulation of the ATP synthesis through conformational changes.

Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the ß-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the ß-hairpin and the redox loop domains specific to CFoCF1.

Changes in intracellular energetic and metabolite states due to increased galactolipid levels in Synechococcus elongatus PCC 7942.

The lipid composition of thylakoid membranes is conserved from cyanobacteria to green plants. However, the biosynthetic pathways of galactolipids, the major components of thylakoid membranes, are known to differ substantially between cyanobacteria and green plants. We previously reported on a transformant of the unicellular rod-shaped cyanobacterium Synechococcus elongatus PCC 7942, namely SeGPT, in which the synthesis pathways of the galactolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol are completely replaced by those of green plants. SeGPT exhibited increased galactolipid content and could grow photoautotrophically, but its growth rate was slower than that of wild-type S. elongatus PCC 7942. In the present study, we investigated pleiotropic effects that occur in SeGPT and determined how its increased lipid content affects cell proliferation. Microscopic observations revealed that cell division and thylakoid membrane development are impaired in SeGPT. Furthermore, physiological analyses indicated that the bioenergetic state of SeGPT is altered toward energy storage, as indicated by increased levels of intracellular ATP and glycogen. We hereby report that we have identified a new promising candidate as a platform for material production by modifying the lipid synthesis system in this way.

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins.

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein-based or yellow fluorescent protein-based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes "ROBINc" and "ROBINy." ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.

The phototroph-specific ß-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria.

The FoF1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FoF1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic ß-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole FoF1 complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire FoF1 ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the ß-hairpin structure of FoF1 ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the ß-hairpin structure, which provided novel insights into the regulatory mechanisms of FoF1 ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.

A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms.

pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as "luminescent pH indicator protein" (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms.

Cytosolic GLUTAMINE SYNTHETASE1;1 Modulates Metabolism and Chloroplast Development in Roots.

Nitrogen (N) is an essential macronutrient, and the final form of endogenous inorganic N is ammonium, which is assimilated by Gln synthetase (GS) into Gln. However, how the multiple isoforms of cytosolic GSs contribute to metabolic systems via the regulation of ammonium assimilation remains unclear. In this study, we compared the effects of two rice (Oryza sativa) cytosolic GSs, namely OsGS1;1 and OsGS1;2, on central metabolism in roots using reverse genetics, metabolomic and transcriptomic profiling, and network analyses. We observed (1) abnormal sugar and organic N accumulation and (2) significant up-regulation of genes associated with photosynthesis and chlorophyll biosynthesis in the roots of Osgs1;1 but not Osgs1;2 knockout mutants. Network analysis of the Osgs1;1 mutant suggested that metabolism of Gln was coordinated with the metabolic modules of sugar metabolism, tricarboxylic acid cycle, and carbon fixation. Transcript profiling of Osgs1;1 mutant roots revealed that expression of the rice sigma-factor (OsSIG) genes in the mutants were transiently upregulated. GOLDEN2-LIKE transcription factor-encoding genes, which are involved in chloroplast biogenesis in rice, could not compensate for the lack of OsSIGs in the Osgs1;1 mutant. Microscopic analysis revealed mature chloroplast development in Osgs1;1 roots but not in the roots of Osgs1;2, Osgs1;2-complemented lines, or the wild type. Thus, organic N assimilated by OsGS1;1 affects a broad range of metabolites and transcripts involved in maintaining metabolic homeostasis and plastid development in rice roots, whereas OsGS1;2 has a more specific role, affecting mainly amino acid homeostasis but not carbon metabolism.

The ß-hairpin region of the cyanobacterial F1-ATPase γ-subunit plays a regulatory role in the enzyme activity.

The γ-subunit of cyanobacterial and chloroplast ATP synthase, the rotary shaft of F1-ATPase, equips a specific insertion region that is only observed in photosynthetic organisms. This region plays a physiologically pivotal role in enzyme regulation, such as in ADP inhibition and redox response. Recently solved crystal structures of the γ-subunit of F1-ATPase from photosynthetic organisms revealed that the insertion region forms a ß-hairpin structure, which is positioned along the central stalk. The structure-function relationship of this specific region was studied by constraining the expected conformational change in this region caused by the formation of a disulfide bond between Cys residues introduced on the central stalk and this ß-hairpin structure. This fixation of the ß-hairpin region in the α3ß3γ complex affects both ADP inhibition and the binding of the ε-subunit to the complex, indicating the critical role that the ß-hairpin region plays as a regulator of the enzyme. This role must be important for the maintenance of the intracellular ATP levels in photosynthetic organisms.

The N-terminal region of the ϵ subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity.

ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ϵ subunits. To clarify the function of the ϵ subunit from phototrophs, here we analyzed the ϵ subunit-mediated inhibition (ϵ-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal α-helix null ϵ-mutants; one lacked the C-terminal α-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two α-helices or an α-helix and a ß-sandwich structure. All of these ϵ-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F1-ATPase in the presence of these ϵ-mutants. The stop angular position of the γ subunit in the presence of the ϵ-mutant was identical to that in the presence of the WT ϵ. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80° forcing of the γ subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ϵ-mutants or by the WT ϵ subunit. These results imply that the C-terminal α-helix domain of the ϵ subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial ϵ, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ϵ subunit-mediated regulation of ATP synthase.

Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.

F1-ATPase forms the membrane-associated segment of F0F1-ATP synthase - the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F1 subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 Šresolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35-40 amino acids. Our structural data reveal that this region forms a ß-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this ß-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F1 subcomplex. Biochemical analyses of the corresponding α3ß3γ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the α3ß3 complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the ß-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the ß-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F1 rotation during ATP hydrolysis.

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase.

F1 is a soluble part of FoF1-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F1 motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α3ß3 hexamer of the F1 molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure-function relationship of the γ subunit, we prepared a mutant F1-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F1-ATP synthase.

Natural Escherichia coli strains undergo cell-to-cell plasmid transformation.

Horizontal gene transfer is a strong tool that allows bacteria to adapt to various environments. Although three conventional mechanisms of horizontal gene transfer (transformation, transduction, and conjugation) are well known, new variations of these mechanisms have also been observed. We recently reported that DNase-sensitive cell-to-cell transfer of nonconjugative plasmids occurs between laboratory strains of Escherichia coli in co-culture. We termed this phenomenon "cell-to-cell transformation." In this report, we found that several combinations of Escherichia coli collection of reference (ECOR) strains, which were co-cultured in liquid media, resulted in DNase-sensitive cell-to-cell transfer of antibiotic resistance genes. Plasmid isolation of these new transformants demonstrated cell-to-cell plasmid transfer between the ECOR strains. Natural transformation experiments, using a combination of purified plasmid DNA and the same ECOR strains, revealed that cell-to-cell transformation occurs much more frequently than natural transformation under the same culture conditions. Thus, cell-to-cell transformation is both unique and effective. In conclusion, this study is the first to demonstrate cell-to-cell plasmid transformation in natural E. coli strains.

Attachment of phycobilisomes in an antenna-photosystem I supercomplex of cyanobacteria.

Oxygenic photosynthesis is driven by photosystems I and II (PSI and PSII, respectively). Both have specific antenna complexes and the phycobilisome (PBS) is the major antenna protein complex in cyanobacteria, typically consisting of a core from which several rod-like subcomplexes protrude. PBS preferentially transfers light energy to PSII, whereas a PSI-specific antenna has not been identified. The cyanobacterium Anabaena sp. PCC 7120 has rod-core linker genes (cpcG1-cpcG2-cpcG3-cpcG4). Their products, except CpcG3, have been detected in the conventional PBS. Here we report the isolation of a supercomplex that comprises a PSI tetramer and a second, unique type of a PBS, specific to PSI. This rod-shaped PBS includes phycocyanin (PC) and CpcG3 (hereafter renamed "CpcL"), but no allophycocyanin or CpcGs. Fluorescence excitation showed efficient energy transfer from PBS to PSI. The supercomplex was analyzed by electron microscopy and single-particle averaging. In the supercomplex, one to three rod-shaped CpcL-PBSs associate to a tetrameric PSI complex. They are mostly composed of two hexameric PC units and bind at the periphery of PSI, at the interfaces of two monomers. Structural modeling indicates, based on 2D projection maps, how the PsaI, PsaL, and PsaM subunits link PSI monomers into dimers and into a rhombically shaped tetramer or "pseudotetramer." The 3D model further shows where PBSs associate with the large subunits PsaA and PsaB of PSI. It is proposed that the alternative form of CpcL-PBS is functional in harvesting energy in a wide number of cyanobacteria, partially to facilitate the involvement of PSI in nitrogen fixation.

Phycobilisome model with novel skeleton-like structures in a glaucocystophyte Cyanophora paradoxa.

Phycobilisome (PBS) is a photosynthetic antenna supercomplex consisting of a central core subcomplex with several peripheral rods radiating from the core. Subunit structure of PBS was studied in a glaucocystophyte Cyanophora paradoxa strain NIES 547. Subunit composition of PBS was identified by N-terminal sequencing and genes for the subunits were determined by homology search of databases. They included rod linker proteins CpcK1 and CpcK2, rod-core linker proteins CpcG1 and CpcG2, and core linker proteins ApcC1 and ApcC2. Subfractionation by native polyacrylamide gel electrophoresis provided evidence for novel subcomplexes (ApcE/CpcK1/CpcG2/ApcA/ApcB/CpcD and ApcE/CpcK2/CpcG1/ApcA/ApcB), which connect rod and core subcomplexes. These skeleton-like structures may serve as a scaffold of the whole PBS assembly. Different roles of ApcC1 and ApcC2 were also suggested. Based on these findings, structural models for PBS were proposed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Distinct roles of CpcG1-phycobilisome and CpcG2-phycobilisome in state transitions in a cyanobacterium Synechocystis sp. PCC 6803.

State transitions in cyanobacteria regulate the relative energy transfer from phycobilisome to photosystem I and II. Although it has been shown that phycobilisome mobility is essential for phycobilisome-dependent state transitions, the biochemical mechanism is not known. Previously we reported that two distinct forms of phycobilisome are assembled with different CpcG copies, which have been referred to as "rod-core linker," in a cyanobacterium Synechocystis sp. PCC 6803. CpcG2-phycobilisome is devoid of a typical central core, while CpcG1-phycobilisome is equivalent to the conventional phycobilisome supercomplex. Here, we demonstrated that the cpcG1 disruptant has a severe specific defect in the phycobilisome-dependent state transition. However, fluorescence recovery after photobleaching measurements showed no obvious difference in phycobilisome mobility between the wild type and the cpcG1 disruptant. This suggests that both CpcG1 and CpcG2 phycobilisomes have an unstable interaction with the reaction centres. However, only CpcG1 phycobilisomes are involved in state transitions. This suggests that state transitions require the phycobilisome core.

The membrane-associated CpcG2-phycobilisome in Synechocystis: a new photosystem I antenna.

The phycobilisome (PBS) is a supramolecular antenna complex required for photosynthesis in cyanobacteria and bilin-containing red algae. While the basic architecture of PBS is widely conserved, the phycobiliproteins, core structure and linker polypeptides, show significant diversity across different species. By contrast, we recently reported that the unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two types of PBSs that differ in their interconnecting "rod-core linker" proteins (CpcG1 and CpcG2). CpcG1-PBS was found to be equivalent to conventional PBS, whereas CpcG2-PBS retains phycocyanin rods but is devoid of the central core. This study describes the functional analysis of CpcG1-PBS and CpcG2-PBS. Specific energy transfer from PBS to photosystems that was estimated for cells and thylakoid membranes based on low-temperature fluorescence showed that CpcG2-PBS transfers light energy preferentially to photosystem I (PSI) compared to CpcG1-PBS, although they are able to transfer to both photosystems. The preferential energy transfer was also supported by the increased photosystem stoichiometry (PSI/PSII) in the cpcG2 disruptant. The cpcG2 disruptant consistently showed retarded growth under weak PSII light, in which excitation of PSI is limited. Isolation of thylakoid membranes with high salt showed that CpcG2-PBS is tightly associated with the membrane, while CpcG1-PBS is partly released. CpcG2 is characterized by its C-terminal hydrophobic segment, which may anchor CpcG2-PBS to the thylakoid membrane or PSI complex. Further sequence analysis revealed that CpcG2-like proteins containing a C-terminal hydrophobic segment are widely distributed in many cyanobacteria.

Distinct roles of CpcG1 and CpcG2 in phycobilisome assembly in the cyanobacterium Synechocystis sp. PCC 6803.

Structural role of the second copy of the rod-core linker CpcG, which was found by genome analysis, was studied in Synechocystis sp. PCC 6803 by gene disruption and fractionation of phycobilisome (sub)complexes. Disruption of cpcG2 (sll1471) resulted in a marked decrease in phycocyanin content both in the background of wild-type and cpcG1 (slr2051)-disruptant. The unique phycocyanin rod-CpcG2 complex without the major allophycocyanin components was isolated from the cpcG1-disruptant. By fluorescence analysis, it was proposed that CpcG2 protein connects the rods with a minor allophycocyanin component, to support energy transfer to Photosystem I.

Complete sequences of six penicillin-binding protein genes from 40 Streptococcus pneumoniae clinical isolates collected in Japan.

All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S. pneumoniae isolates collected in Japan, were completely sequenced. The MICs of penicillin for these strains varied between 0.015 and 8 microg/ml. In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains. These six strains were highly resistant (MICs >/= 2 microg/ml) to cefotaxime. The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains. In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 microg/ml) was the highest imipenem MIC detected. In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were >/=1 microg/ml. In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 microg/ml) was the highest cefotaxime MIC detected. On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed. The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S. pneumoniae resistance to beta-lactam antibiotics. Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with beta-lactam resistance.