Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus(MRSA) (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs by virtue of their genomic background and increased virulence in animal models. Here, we show that in two common CA-MRSA isolates, USA300 and MW2 (USA400), a loss of penicillin binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance, thus demonstrating that mecA, encoding PBP2A, is not the sole determinant of methicillin resistance in CA-MRSA. The loss of PBP4 was also found to severely affect the transcription of PBP2 in cells after challenge with oxacillin, thus leading to a significant decrease in peptidoglycan cross-linking. Autolysis, which is commonly associated with the killing mechanism of penicillin and ␤-lactams, does not play a role in the reduced resistance phenotype associated with the loss of PBP4. We also showed that cefoxitin, a semisynthetic ␤-lactam that binds irreversibly to PBP4, is synergistic with oxacillin in killing CA-MRSA strains, including clinical CA-MRSA isolates. Thus, PBP4 represents a major target for drug rediscovery against CA-MRSA, and a combination of cefoxitin and synthetic penicillins may be an effective therapy for CA-MRSA infections.

Bacterial toxin-antitoxin (TA) systems typically consist of a small, labile antitoxin that inactivates a specific longer-lived toxin. In Escherichia coli, such antitoxins are proteolytically regulated by the ATP-dependent proteases Lon and ClpP. Under normal conditions, antitoxin synthesis is sufficient to replace this loss from proteolysis, and the bacterium remains protected from the toxin. However, if TA production is interrupted, antitoxin levels decrease, and the cognate toxin is free to inhibit the specific cellular component, such as mRNA, DnaB, or gyrase. To date, antitoxin degradation has been studied only in E. coli, so it remains unclear whether similar mechanisms of regulation exist in other organisms. To address this, we followed antitoxin levels over time for the three known TA systems of the major human pathogen Staphylococcus aureus, mazEF, axe1-txe1, and axe2-txe2. We observed that the antitoxins of these systems, MazE sa , Axe1, and Axe2, respectively, were all degraded rapidly (half-life [t 1/2 ], ϳ18 min) at rates notably higher than those of their E. coli counterparts, such as MazE (t 1/2 , ϳ30 to 60 min). Furthermore, when S. aureus strains deficient for various proteolytic systems were examined for changes in the half-lives of these antitoxins, only strains with clpC or clpP deletions showed increased stability of the molecules. From these studies, we concluded that ClpPC serves as the functional unit for the degradation of all known antitoxins in S. aureus.Staphylococcus aureus is a versatile human pathogen responsible for an increasing number of hospital-and community-acquired infections (33, 41) ranging from superficial skin lesions to life-threatening sepsis, endocarditis, and toxic shock (29). S. aureus' capacity to cause illness is enhanced by its robust stress response, which allows it to endure adverse conditions, such as heat, antibiotics, and nutritional deprivation. This is mediated in part by transcriptional regulators, like CtsR (11), CodY (31), and the alternative sigma factor B (24), that allow the bacteria to rapidly adjust to challenging environments.The roles of chromosomal toxin-antitoxin (TA) modules in environmental and antibiotic stress response have been documented for a variety of organisms, especially Escherichia coli, but only recently have they been investigated in S. aureus (12,18,43). These systems typically consist of a pair of cotranscribed stress-inducible genes (19) that encode a stable toxin and a more labile antitoxin. Depletion of the antitoxin allows activation of its cognate toxin, which is then free to interfere with a specific cellular target, such as mRNA, DNA gyrase, or DNA helicase. Depending on the species and the TA system, this activation results in a variety of phenotypes, but those related to growth, stress response, starvation, and persistence are often seen (12,19,30). For example, Streptococcus mutans devoid of its TA systems is more susceptible to changes in nutrient availability and pH than its counterpart wild-type strains (26). Further...

The role of chromosomally encoded toxin-antitoxin (TA) loci in bacterial physiology has been under debate, with the toxin proposed as either an inducer of bacteriostasis or a mediator of programmed cell death (PCD). We report here that ectopic expression of MazF Sa , a toxin of the TA module from Staphylococcus aureus, led to a rapid decrease in CFU counts but most cells remained viable as determined by differential Syto 9 and propidium iodide staining after MazF Sa induction. This finding suggested that the toxin MazF Sa induced cell stasis rather than cell death. We also showed that MazF Sa selectively cleaves cellular mRNAs in vivo, avoiding "important" transcripts such as recA, gyrB, and sarA mRNAs in MazF Sa -induced cells, while these three mRNAs can be cleaved in vitro. The results of Northwestern blotting showed that both sarA and recA mRNAs bind strongly to a putative RNA-binding protein. These data suggest that S. aureus likely undergoes stasis by protecting selective mRNA with RNA-binding proteins upon the expression of MazF Sa in vivo.Many bacteria have chromosomally encoded toxin-antitoxin (TA) loci in which the toxin and antitoxin genes exist in an operon and are coexpressed to form a TA complex. The toxin is stable, while the antitoxin is labile and can be degraded in vivo by host proteases (e.g., ClpP or Lon in Escherichia coli). Under conditions of stress whereby transcription of the TA operon is repressed and which hence preclude the continuous synthesis of the labile antitoxin, the more-stable toxin can unleash its toxic effect to inhibit cell growth. However, metabolic stresses, such as amino acid and carbon source starvation, have been shown to induce transcription of E. coli mazEF and other TA loci in E. coli (7,9,14). Studies with several toxin systems indicate that many toxins are probably sequence-specific endoribonucleases. For instance, MazF of E. coli cleaves mRNA at ACA sites both in vitro and in vivo (30), while the RelE toxin, also from E. coli, cleaves mRNA positioned at the ribosomal A site both in vitro and in vivo, with cleavage occurring between the second and third bases of the A site codon

The mazEF homologs of Staphylococcus aureus, designated mazEF sa , have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF Sa , as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF Sa leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF Sa is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF Sa cleaves single-strand RNA preferentially at the 5 side of the first U or 3 side of the second U residue within the consensus sequences VUUV (where V and V are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE Sa binds MazF Sa to form a complex to inhibit the endoribonuclease activity of MazF Sa . Contrary to the system in E. coli, exposure to selected antibiotics augmented mazEF sa transcription, akin to what one would anticipate from the environmental stress response of the sigB system. These data indicate that the mazEF system of S. aureus differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.

BackgroundScale-up to industrial production level of a fermentation process occurs after optimization at small scale, a critical transition for successful technology transfer and commercialization of a product of interest. At the large scale a number of important bioprocess engineering problems arise that should be taken into account to match the values obtained at the small scale and achieve the highest productivity and quality possible. However, the changes of the host strain’s physiological and metabolic behavior in response to the scale transition are still not clear.ResultsHeterogeneity in substrate and oxygen distribution is an inherent factor at industrial scale (10,000 L) which affects the success of process up-scaling. To counteract these detrimental effects, changes in dissolved oxygen and pressure set points and addition of diluents were applied to 10,000 L scale to enable a successful process scale-up. A comprehensive semi-quantitative and time-dependent analysis of the exometabolome was performed to understand the impact of the scale-up on the metabolic/physiological behavior of the host microorganism. Intermediates from central carbon catabolism and mevalonate/ergosterol synthesis pathways were found to accumulate in both the 10 L and 10,000 L scale cultures in a time-dependent manner. Moreover, excreted metabolites analysis revealed that hypoxic conditions prevailed at the 10,000 L scale. The specific product yield increased at the 10,000 L scale, in spite of metabolic stress and catabolic-anabolic uncoupling unveiled by the decrease in biomass yield on consumed oxygen.ConclusionsAn optimized S. cerevisiae fermentation process was successfully scaled-up to an industrial scale bioreactor. The oxygen uptake rate (OUR) and overall growth profiles were matched between scales. The major remaining differences between scales were wet cell weight and culture apparent viscosity. The metabolic and physiological behavior of the host microorganism at the 10,000 L scale was investigated with exometabolomics, indicating that reduced oxygen availability affected oxidative phosphorylation cascading into down- and up-stream pathways producing overflow metabolism. Our study revealed striking metabolic and physiological changes in response to hypoxia exerted by industrial bioprocess up-scaling.

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Various approaches have been applied to optimize biological product fermentation processes and define design space. In this article, we present a stepwise approach to optimize a Saccharomyces cerevisiae fermentation process through risk assessment analysis, statistical design of experiments (DoE), and multivariate Bayesian predictive approach. The critical process parameters (CPPs) were first identified through a risk assessment. The response surface for each attribute was modeled using the results from the DoE study with consideration given to interactions between CPPs. A multivariate Bayesian predictive approach was then used to identify the region of process operating conditions where all attributes met their specifications simultaneously. The model prediction was verified by twelve consistency runs where all batches achieved broth titer more than 1.53 g/L of broth and quality attributes within the expected ranges. The calculated probability was used to define the reliable operating region. To our knowledge, this is the first case study to implement the multivariate Bayesian predictive approach to the process optimization for the industrial application and its corresponding verification at two different production scales. This approach can be extended to other fermentation process optimizations and reliable operating region quantitation.

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