The Staphylococcus aureus Methicillin Resistance Factor FmtA Is a d-Amino Esterase That Acts on Teichoic Acids.

UNLABELLED: The methicillin resistance factor encoded by fmtA is a core member of the Staphylococcus aureus cell wall stimulon, but its function has remained elusive for the past two decades. First identified as a factor that affects methicillin resistance in S. aureus strains, FmtA was later shown to interact with teichoic acids and to localize to the cell division septum. We have made a breakthrough in understanding FmtA function. We show that FmtA hydrolyzes the ester bond between d-Ala and the backbone of teichoic acids, which are polyglycerol-phosphate or polyribitol-phosphate polymers found in the S. aureus cell envelope. FmtA contains two conserved motifs found in serine active-site penicillin-binding proteins (PBPs) and ß-lactamases. The conserved SXXK motif was found to be important for the d-amino esterase activity of FmtA. Moreover, we show that deletion of fmtA (ΔfmtA) led to higher levels of d-Ala in teichoic acids, and this effect was reversed by complementation of ΔfmtA with fmtA. The positive charge on d-Ala partially masks the negative charge of the polyol-phosphate backbone of teichoic acids; hence, a change in the d-Ala content will result in modulation of their charge. Cell division, biofilm formation, autolysis, and colonization are among the many processes in S. aureus affected by the d-Ala content and overall charge of the cell surface teichoic acids. The esterase activity of FmtA and the regulation of fmtA suggest that FmtA functions as a modulator of teichoic acid charge, thus FmtA may be involved in S. aureus cell division, biofilm formation, autolysis, and colonization. IMPORTANCE: Teichoic acids are involved in cell division, cell wall synthesis, biofilm formation, attachment of bacteria to artificial surfaces, and colonization. However, the function of teichoic acids is not fully understood. Modification by glycosylation and/or d-alanylation of the polyol-phosphate backbone of teichoic acids is important in the above cell processes. The intrinsic negative charge of teichoic acid backbone plays a role in the charge and/or pH of the bacterial surface, and d-alanylation represents a means through which bacteria modulate the charge or the pH of their surfaces. We discovered that FmtA removes d-Ala from teichoic acids. We propose FmtA may provide a temporal and spatial regulation of the bacterial cell surface charge in two ways, by removing the d-Ala from LTA to make it available to wall teichoic acid (WTA) in response to certain conditions and by removing it from WTA to allow the cell to reset its surface charge to a previous condition.

Staphylococcus aureus methicillin-resistance factor fmtA is regulated by the global regulator SarA.

fmtA encodes a low-affinity penicillin binding protein in Staphylococcus aureus. It is part of the core cell wall stimulon and is involved in methicillin resistance in S. aureus. Here, we report that the transcription factor, SarA, a pleiotropic regulator of virulence genes in S. aureus, regulates the expression of fmtA. In vitro binding studies with purified SarA revealed that it binds to specific sites within the 541-bp promoter region of fmtA. Mutation of a key residue of the regulatory activity of SarA (Arg90) abolished binding of SarA to the fmtA promoter, suggesting that SarA binds specifically to the fmtA promoter region. In vivo analysis of the fmtA promoter using a lux operon reporter fusion show high level expression following oxacillin induction, which was abrogated in a sarA mutant strain. These data suggest that SarA is essential for the induction of fmtA expression by cell wall-specific antibiotics. Further, in vitro transcription studies show that SarA enhances fmtA transcription and suggest that regulation of fmtA could be via a SigA-dependent mechanism. Overall, our results show that SarA plays a direct role in the regulation of fmtA expression via binding to the fmtA promoter.

Delay in virus accumulation and low virus transmission from transgenic rice plants expressing Rice tungro spherical virus RNA.

Rice tungro, a devastating viral disease of rice in South and Southeast Asia, is caused by the joint infection of a DNA virus, Rice tungro bacilliform virus (RTBV) and an RNA virus Rice tungro spherical virus (RTSV). RTBV and RTSV are transmitted exclusively by the insect vector Green leafhopper (GLH). RTSV is necessary for the transmission of RTBV. To obtain transgenic resistance against RTSV, indica rice plants were transformed using DNA constructs designed to express an untranslatable sense or anti-sense RTSV RNA. Progeny of primary transformants showing low copies of the integrated transgenes and accumulating the corresponding transcripts at low levels were challenged with viruliferous GLH. Three out of four transgenic plant lines expressing untranslatable RTSV RNA in the sense orientation and two out of the four lines expressing an RTSV gene in the anti-sense orientation showed delayed buildup of RTSV RNA over time. Transmission of RTBV from the above lines was reduced significantly.

Roles of DNA sequence and sigma A factor in transcription of the vraSR operon.

Cell wall damage in Staphylococcus aureus induces a rapid genome-wide response, referred to as the cell wall stress stimulon. This response is mediated by a two-component system, the vancomycin resistance-associated sensor/regulator (VraSR). The response regulator protein VraR is a transcription factor. Here, we demonstrate that two VraR binding sites in the vraSR operon control region are involved in the regulation of the vraSR operon. The sites are centered at the -60 and -35 nucleotide positions and are referred to as R1 and R2, respectively. DNase I footprinting and lux operon reporter vector studies showed that both of these sites communicate intimately with each other to fine-tune the activity of the vraSR operon. Mutagenesis of the VraR binding sites showed that dimerization of unphosphorylated VraR at R1 is driven by a hierarchy in VraR binding and by the proximity of the two tandem VraR binding sequences at this site. On the other hand, these studies show that the lack of sequence conservation and the distance between the VraR binding sequences in R2 ensure that VraR is recruited to this site only when phosphorylated (hence, under stress conditions). Furthermore, we demonstrate that sigma A (SigA) factor is involved in the regulation of the vraSR operon. Our study shows that sigma A factor does not bind to the vraSR operon control region in the absence of VraR, suggesting that VraR may interact directly with this factor.

Hydrolytic mechanism of OXA-58 enzyme, a carbapenem-hydrolyzing class D ß-lactamase from Acinetobacter baumannii.

Carbapenem-hydrolyzing class D ß-lactamases (CHDLs) represent an emerging antibiotic resistance mechanism encountered among the most opportunistic Gram-negative bacterial pathogens. We report here the substrate kinetics and mechanistic characterization of a prominent CHDL, the OXA-58 enzyme, from Acinetobacter baumannii. OXA-58 uses a carbamylated lysine to activate the nucleophilic serine used for ß-lactam hydrolysis. The deacylating water molecule approaches the acyl-enzyme species, anchored at this serine (Ser-83), from the α-face. Our data show that OXA-58 retains the catalytic machinery found in class D ß-lactamases, of which OXA-10 is representative. Comparison of the homology model of OXA-58 and the recently solved crystal structures of OXA-24 and OXA-48 with the OXA-10 crystal structure suggests that these CHDLs have evolved the ability to hydrolyze imipenem, an important carbapenem in clinical use, by subtle structural changes in the active site. These changes may contribute to tighter binding of imipenem to the active site and removal of steric hindrances from the path of the deacylating water molecule.

Virus-induced gene silencing in rice using a vector derived from a DNA virus.

Virus-induced gene silencing (VIGS) is a method of rapid and transient gene silencing in plants using viral vectors. A VIGS vector for gene silencing in rice has been developed from Rice tungro bacilliform virus (RTBV), a rice-infecting virus containing DNA as the genetic material. A full-length RTBV DNA cloned as a partial dimer in a binary plasmid accumulated in rice plants when inoculated through Agrobacterium (agroinoculation) within 2 weeks and produced detectable levels of RTBV coat protein. Deletion of two of the four viral ORFs did not compromise the ability of the cloned RTBV DNA to accumulate in rice plants. To modify the cloned RTBV DNA as a VIGS vector (pRTBV-MVIGS), the tissue-specific RTBV promoter was replaced by the constitutively expressed maize ubiquitin promoter, sequences comprising the tRNA-binding site were incorporated to ensure reverse transcription-mediated replication, sequences to ensure optimal context for translation initiation of the viral genes were added and a multi-cloning site for the ease of cloning DNA fragments was included. The silencing ability of pRTBV-MVIGS was tested using the rice phytoene desaturase (pds) gene on rice. More than half of the agroinoculated rice plants showed white streaks in leaves within 21 days post-inoculation (dpi), which continued to appear in all emerging leaves till approximately 60-70 dpi. Compared to control samples, real-time PCR showed only 10-40% accumulation of pds transcripts in the leaves showing the streaks. This is the first report of the construction of a VIGS vector for rice which can be introduced by agroinoculation.

DNA-binding activity of the vancomycin resistance associated regulator protein VraR and the role of phosphorylation in transcriptional regulation of the vraSR operon.

In Staphylococcus aureus the VraSR two-component system acts as a sentinel that can rapidly sense cell wall peptidoglycan damage and coordinate a response to enhance the resistance phenotype. VraR is a transcription factor and its cognate kinase, VraS, modulates the DNA-binding activity of VraR by regulating its phosphorylation state and hence its dimerization state. Here we provide the first report on the VraR transcriptional activity by investigating the interaction with the vraSR operon control region. We found that this region contains three VraR-binding sites, each with unique VraR-binding features. VraR binding to the most conserved site is phosphorylation independent, and dimerization is proposed to be induced through binding to DNA. By contrast, binding to the less conserved site requires phosphorylation of VraR. This site overlaps with the binding site of the sigma subunit of the RNA polymerase complex, suggesting that VraR could be interacting with the transcription machinery in the presence of the cell wall stress signal. Mutagenesis studies on the VraR binding sites suggest that there is directionality in the binding of VraR to the target DNA, probably dictated by VraR dimerization. We also constructed a P(vraSR)-fused lux operon reporter vector to investigate in vivo the significance of our in vitro studies. These studies show that upon cell wall stress, induced by oxacillin, the expression level of the lux operon goes up and it is affected by the integrity of the two identified VraR-binding sites in agreement with the in vitro studies. Further, they demonstrate that the VraR most conserved binding site is essential to the vraSR operon expression. On the other hand, they suggest that the role of the VraR less conserved site could be that of mediating high levels of vraSR operon expression during cell wall stress conditions.

PHOTOSYNTHETIC INORGANIC CARBON ACQUISITION IN AN ACID-TOLERANT, FREE-LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)(1).

The processes of CO2 acquisition were characterized for the acid-tolerant, free-living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO3 (-) uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO2 formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO2 uptake and O2 evolution by membrane-inlet mass spectrometry demonstrated that air-grown cells reduced the CO2 concentration in the medium to an equilibrium concentration of 15 µM, but AZA-treated cells caused a drop in extracellular CO2 concentration to a compensation concentration of 27 µM at pH 8.0. CO2 -pulsing experiments with cells in the light indicated that the cells do not actively take up CO2 . An internal pool of unfixed inorganic carbon was not detected at the CO2 compensation concentration, probably because of the lack of active CO2 uptake, but was detectable at times before compensation point was reached. These results indicate that this free-living Coccomyxa possesses a CO2 -concentrating mechanism (CCM) due to an active bicarbonate-uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.