Ana Madalena Ludovice

Extensive and Genome-Wide Changes in the Transcription Profile of Staphylococcus aureus Induced by Modulating the Transcription of the Cell Wall Synthesis Gene murF

A murF conditional mutant was used to evaluate the effect of suboptimal transcription of this gene on the transcriptome of the methicillin-resistant Staphylococcus aureus strain COL. The mutant was grown in the presence of optimal and suboptimal concentrations of the inducer, and the relative levels of transcription of genes were evaluated genome wide with an Affymetrix DNA microarray that included all open reading frames (ORFs) as well as intergenic sequences derived from four sequenced S. aureus strains. Using a sensitivity threshold value of 1.5, suboptimal expression of murF altered the transcription of a surprisingly large number of genes, i.e., 668 out of the 2,740 ORFs (close to one-fourth of all ORFs), of the genome of S. aureus strain COL. The genes with altered transcription were distributed evenly around the S. aureus chromosome, and groups of genes involved with distinct metabolic functions responded in unique and operon-specific manners to modulation in murF transcription. For instance, all genes belonging to the isd operon and all but 2 of the 35 genes of prophage L54a were down-regulated, whereas all but one of the 21 members of the vraSR regulon and most of the 79 virulence-related genes (those for fibronectin binding proteins A and B, clumping factor A, gamma hemolysin, enterotoxin B, etc.) were up-regulated in cells with suboptimal expression of murF. Most importantly, the majority of these altered gene expression profiles were reversible by resupplying the optimal concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) to the culture. The observations suggest the coordinate regulation of a large sector of the S. aureus transcriptome in response to a disturbance in cell wall synthesis.

Identification of genetic determinants and enzymes involved with the amidation of glutamic acid residues in the peptidoglycan of Staphylococcus aureus.

The glutamic acid residues of the peptidoglycan of Staphylococcus aureus and many other bacteria become amidated by an as yet unknown mechanism. In this communication we describe the identification, in the genome of S. aureus strain COL, of two co-transcribed genes, murT and gatD, which are responsible for peptidoglycan amidation. MurT and GatD have sequence similarity to substrate-binding domains in Mur ligases (MurT) and to the catalytic domain in CobB/CobQ-like glutamine amidotransferases (GatD). The amidation of glutamate residues in the stem peptide of S. aureus peptidoglycan takes place in a later step than the cytoplasmic phase – presumably the lipid phase - of the biosynthesis of the S. aureus cell wall precursor. Inhibition of amidation caused reduced growth rate, reduced resistance to beta-lactam antibiotics and increased sensitivity to lysozyme which inhibited culture growth and caused degradation of the peptidoglycan.

Normally Functioning murF Is Essential for the Optimal Expression of Methicillin Resistance in Staphylococcus aureus

A carboxy-terminal fragment of murF was used to construct and insert a suicide plasmid into the chromosomal copy of the gene in the highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain COL by Campbell type integration. The plasmid insertion generated a mutant in which the MIC value for oxacillin was reduced from 400 microg/ml of the parental strain to 0.75 microg/ml in 90% of the cells of the mutant cultures that were heterogeneous: they contained subpopulations of bacteria with a frequency of 10(-3) that were capable of expressing resistance at nearly the parental level. The impact of the murF mutation on antibiotic resistance was selective for beta-lactam antibiotics: there was no change in the susceptibility of the mutant to D-cycloserine, fosfomycin, beta-D-chloro-alanine, moenomycin, bacitracin, or vancomycin. Analysis of the mutant peptidoglycan showed decrease in the percentage of oligomeric components in rough proportion to the accumulation of several abnormal muropeptide components, which were identified as structural variants of the disaccharide tripeptide monomer. An abnormal cell wall precursor identified as UDP MurNac tripeptide was also detected in the cytoplasmic pool of the mutant strain. A normal proportion of oligomers and a greatly reduced representation of the disaccharide tripeptide were demonstrated in the cell wall of the murF mutants subpopulation that has retained the parental level of resistance. Northern analysis demonstrated a drastic reduction in the transcription rate of mecA in mutant F9 whereas mecA transcription increased in the subpopulation of bacteria that retained high-level resistance.

Role of murE in the Expression of β-Lactam Antibiotic Resistance in Staphylococcus aureus

It was shown earlier that Tn551 inserted into the C-terminal region of murE of parental methicillin-resistant Staphylococcus aureus strain COL causes a drastic reduction in methicillin resistance, accompanied by accumulation of UDP-MurNAc dipeptide in the cell wall precursor pool and incorporation of these abnormal muropeptides into the peptidoglycan of the mutant. Methicillin resistance was recovered in a suppressor mutant. The murE gene of the same strain was then put under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter P(spac). Bacteria grown in the presence of suboptimal concentrations of IPTG accumulated UDP-MurNAc dipeptide in the cell wall precursor pool. Both growth rates and methicillin resistance levels (but not resistance to other antibiotics) were a function of the IPTG concentration. Northern analysis showed a gradual increase in the transcription of murE and also in the transcription of pbpB and mecA, parallel with the increasing concentrations of IPTG in the medium. A similar increase in the transcription of pbpB and mecA, the structural genes of penicillin-binding protein 2 (PBP2) and PBP2A, was also detected in the suppressor mutant. The expression of these two proteins, which are known to play critical roles in the mechanism of staphylococcal methicillin resistance, appears to be-directly or indirectly-under the control of the murE gene. Our data suggest that the drastic reduction of the methicillin MIC seen in the murE mutant may be caused by the insufficient cellular amounts of these two PBPs.

Role of murF in Cell Wall Biosynthesis: Isolation and Characterization of a murF Conditional Mutant of Staphylococcus aureus

The Staphylococcus aureus murF gene was placed under the control of a promoter inducible by IPTG (isopropyl-beta-d-thiogalactopyranoside). It was demonstrated that murF is an essential gene; it is cotranscribed with ddlA and growth rate, level of beta-lactam antibiotic resistance, and rates of transcription of the mecA and pbpB genes paralleled the rates of transcription of murF. At suboptimal concentrations of the inducer, a UDP-linked muramyl tripeptide accumulated in the cytoplasm in parallel with the decline in the amounts of the normal pentapeptide cell wall precursor. The abnormal tripeptide component incorporated into the cell wall as a monomeric muropeptide, accompanied by a decrease in the oligomerization degree of the peptidoglycan. However, incorporation of the tripeptide into the cell wall was limited to a relatively low threshold value. Further reduction of the amounts of pentapeptide cell wall precursor caused a gradual decrease in the cellular amounts of peptidoglycan, the production of a thinner peripheral cell wall, aberrant septae, and an overall increase in the diameter of the cells. The observations suggest that the role of murF exceeds its primary function in peptidoglycan biosynthesis and may also be involved in the control of cell division.

Application of Molecular Typing Methods to Characterize Nosocomial Coagulase-Negative Staphylococci Collected in a Greek Hospital During a Three-Year Period (1998–2000)

A total of 143 methicillin-resistant coagulase-negative staphylococci (MR-CNS) collected between 1998 and 2000 at the University Hospital of Patras, Greece, were characterized by antibiogram and genomic typing to define the clonal types endemic in this hospital and their evolution during the 3-year period. These isolates corresponded to 93 methicillin-resistant Staphylococcus epidermidis (MRSE) and 50 other MR-CNS, which were isolated from patients in different wards, exclusively from blood and catheter tips cultures. Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with mecA and murE DNA probes were performed. The application of these methodologies demonstrated the existence, persistence and spread of MRSE, MR-Staphylococcus haemolyticus, and MR-Staphylococcus hominis clones in this hospital, whereas the SmaI/murE hybridization pattern was shown to be a valuable tool for the MRSE identification.

The glucosaminidase domain of Atl – the major Staphylococcus aureus autolysin – has DNA-binding activity

In this communication, we describe evidence demonstrating the capacity of Atl, the major Staphylococcus aureus autolytic enzyme to bind DNA. Electrophoretic mobility shift assays (EMSA) show that both the Atl protein and the endo‐β‐N‐acetylglucosaminidase (GL) domain were able to bind DNA of nonspecific sequence. The implications of this unexpected observation for the physiology of S. aureus remain to be explored.

Contribution of Peptidoglycan Amidation to Beta-Lactam and Lysozyme Resistance in Different Genetic Lineages of Staphylococcus aureus

The enzymes responsible for peptidoglycan amidation in Staphylococcus aureus, MurT and GatD, were recently identified and shown to be required for optimal expression of resistance to beta-lactams, bacterial growth, and resistance to lysozyme. In this study, we analyzed the impact of peptidoglycan amidation in representative strains of the most widespread clones of methicillin resistant S. aureus (MRSA). The inhibition of the expression of murT-gatD operon resulted in different phenotypes of resistance to beta-lactams and lysozyme according to the different genetic backgrounds. Further, clonal lineages CC1 and CC398 (community-acquired MRSA [CA-MRSA]) showed a stronger dependency on MurT-GatD for resistance to beta-lactams, when compared to the impact of the impairment of the cell wall step catalyzed by MurF. In the remaining backgrounds similar phenotypes of beta-lactam resistance were observed upon the impairment of both cell-wall-related genes. Therefore, for CA-related backgrounds, the predominant beta-lactam resistance mechanism seems to involve genes associated with secondary modifications of peptidoglycan. On the other hand, the lack of glutamic acid amidation had a more substantial impact on lysozyme resistance for cells of CA-MRSA backgrounds, than for hospital-acquired MRSA (HA-MRSA). However, no significant differences were found in the resistance level of the respective peptidoglycan structure, suggesting that the lysozyme resistance mechanism involves other factors. Taken together, these results suggested that the different genetic lineages of MRSA were able to develop different molecular strategies to overcome the selective pressures experienced during evolution.

Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.