Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to beta-lactam antibiotics. PBP2A crosslinks nascent peptidoglycan when the native TPs are inhibited by beta-lactams. Although mecA expression is essential for beta-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes MRSA strains to beta-lactams even though the beta-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and beta-lactams is noteworthy not simply because strategies to overcome methicillin-resistant S. aureus (MRSA) are desperately needed, but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The “synthetic lethality” of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic resistant bacterial infections.

The molecular events following inhibition of bacterial peptidoglycan synthesis have not been studied extensively. Previous proteomic studies have revealed that certain proteins are produced in increased amounts upon challenge of Staphylococcus aureus with cell-wall-active antibiotics.In an effort to further those studies, the genes upregulated in their expression in response to cell-wall-active antibiotics have been identified by genome-wide transcriptional profiling using custom-made Affymetrix S. aureus GeneChips TM . A large number of genes, including ones encoding proteins involved in cell-wall metabolism (including pbpB, murZ, fmt and vraS) and stress responses (including msrA, htrA, psrA and hslO), were upregulated by oxacillin, D-cycloserine or bacitracin. This response may represent the transcriptional signature of a cell-wall stimulon induced in response to cell-wall-active agents. The findings imply that treatment with cell-wall-active antibiotics results in damage to proteins including oxidative damage. Additional genes in a variety of functional categories were upregulated uniquely by each of the three cell-wall-active antibiotics studied. These changes in gene expression can be viewed as an attempt by the organism to defend itself against the antibacterial activities of the agents.

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Choline, glycine betaine, and L-proline enhanced the growth of Staphylococcus aureus at high osmolarity (i.e., they acted as osmoprotectants) on various liquid and solid defined media, while an osmoprotective effect of taurine was shown only for cells growing on high-NaCl solid medium that lacked other osmoprotectants. Potassium pool levels were high, and there was little difference in levels in cells grown at different osmolarities. Glycine betaine accumulated to high levels in osmotically stressed cells, and choline was converted to glycine betaine. Proline and taurine also accumulated in response to osmotic stress but to lower levels than glycine betaine.

Branched-chain fatty acids (BCFAs) typically constitute more than 90 % of the fatty acids of Listeria monocytogenes. The authors have previously described two Tn917-induced, cold-sensitive, BCFA-deficient (<40 %) L. monocytogenes mutants (cld-1 and cld-2) with lowered membrane fluidity. Sequence analyses revealed that Tn917 was inserted into different genes of the branched-chain a-keto acid dehydrogenase cluster (bkd) in these two mutants. The cold-sensitivity and BCFA deficiency of cld-1, in which Tn917 was inserted into bkdB, were complemented in trans by cloned bkdB. The growth and corresponding BCFA content of the mutants at 37 6C were stimulated by fatty acid precursors bypassing Bkd, 2-methylbutyrate (precursor for odd-numbered anteiso-fatty acids), isobutyrate (precursor for even-numbered iso-fatty acids) and isovalerate (precursor for odd-numbered iso-fatty acids). In contrast, the corresponding Bkd substrates, a-ketomethylvalerate, a-ketoisovalerate and a-ketoisocaproate, exhibited much poorer activity. At 26 6C, 2-methylbutyrate and isovalerate stimulated the growth of the mutants, and at 10 6C, only 2-methylbutyrate stimulated growth. Pyruvate depressed the BCFA content of cld-2 from 33 % to 27 %, which may be close to the minimum BCFA requirement for L. monocytogenes. The transcription of bkd was enhanced by Bkd substrates, but not by low temperature. When provided with the BCFA precursors, cld-2 was able to increase its anteiso-C 15 : 0 fatty acid content at 10 6C compared to 37 6C, which is the characteristic response of L. monocytogenes to low temperature. This implies that Bkd is not the major cold-regulation point of BCFA synthesis. INTRODUCTIONListeria monocytogenes causes the potentially life-threatening infection known as listeriosis, which has a fatality rate of 20-25 % (Mead et al., 1999). Listeriosis outbreaks have been linked to the consumption of food contaminated with this organism. Nowadays, there is an increased consumption of ready-to-eat, chilled and frozen foods, with decreased use of chemical microbial control agents. The ability of L. monocytogenes to grow at refrigeration temperatures is a critical aspect of its role as a food-borne pathogen, since refrigeration temperature inhibits the growth of most other food-borne pathogens (Bryan, 2004). However, the underlying psychrotolerance mechanism is not completely understood, and a better understanding may result in a novel strategy for controlling the growth of L. monocytogenes at low temperatures.Low temperature has profound effects on all aspects of microbial cell structure and function, involving the structural integrity of macromolecules, macromolecular assemblies, protein synthesis and nutrient uptake (Panoff et al., 1998;Weber & Marahiel, 2002 Two Tn917 transposon-induced mutants (cld-1 and cld-2) have lost the ability to grow at 4 u C on solid media, but are not defective in the induction of cold-shock proteins (Bayles et al., 1996). These mutants are deficient in the production of odd-numbered BCFAs, and exhibit atypical am...