MazEF-rifampicin Interaction Suggests a Mechanism for Rifampicin Induced Inhibition of Persisters

Background: Persistence is a natural phenomenon whereby a subset of a population of isogenic bacteria either grow slow or become dormant conferring them with the abilityto withstand various stresses including antibiotics.In a clinical setting bacterial persistence often leads to the recalcitrance of various infectionsincreasing the treatment time and costs.Additionally, some studies also indicate that persistence can also pave way for the emergence of resistant strains. In a laboratory setting this persistent phenotype is enriched in nutritionally deprived environments. Consequently, in a batch culture the late stationary phase is enriched with persistent bacteria. The mechanism of persister cell formation and its regulation is not well understood. Toxin-antitoxins (TA) systems have been implicated to be responsible for bacterial persistence and rifampicin is used to treat highly persistent bacterial strains. The current study tries to explore a possible interaction between rifampicin and the MazEFTA system that furthers the former’s success rate in treating persistent bacteria.Results: In the current study we found that the population of bacteria in the death phase of a batch cultureconsists of metabolically inactive live cells resembling persisters, which showed higher membrane depolarization as compared to the log phase bacteria. We also observed an increase in the expression of the MazEF TA modules in this phase. Since rifampicin is commonly used to kill the persisters, we assessed the interaction of rifampicin with MazEF complex. We showed that rifampicin directly interacts with MazEF complex.Conclusion: Our study suggests that the interaction of rifampicin with MazEF complex might play an important role in inhibition of persisters.

Investigating the Effects of Osmolytes and Environmental pH on Bacterial Persisters

ABSTRACT Bacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance toward antibiotics. Persisters have been linked to the recalcitrance of biofilm-related infections; hence, a complete understanding of their physiology can lead to improvement of therapeutic strategies for such infections. Mechanisms pertaining to persister formation are thought to be associated with stress response pathways triggered by intra- or extracellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH-induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, we studied the effects of various osmolytes and pH conditions on Escherichia coli persistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite, and acidic pH, significantly reduced persister formation in E. coli compared to no-osmolyte/no-buffer controls, this reduction in persister levels was less pronounced in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

Investigating the Effects of Osmolytes and Environmental pH on Bacterial Persisters

ABSTRACTBacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance towards antibiotics. Persisters have been linked to the recalcitrance of biofilm related infections; hence, a complete understanding of the physiology of persisters can lead to improvement of therapeutic strategies associated with such infections. Mechanisms pertaining to persister formation are known to be related to stress response pathways triggered from intra- or extra-cellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH- induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, here we studied the effects of various osmolytes and pH conditions onEscherichia colipersistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite and acidic pH, significantly reduced persister formation inE. colicompared to no-osmolyte/no-buffer controls, this reduction in persister levels was lessened in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

SCREENING OF TOXIC MARINE NITZSCHIA SPECIES (BACILLARIOPHYCEAE) IN MALAYSIA

<p>Amnesic Shellfish Poisoning (ASP) is a type of intoxication caused by the neurotoxin domoic acid (DA). The diatom genus <em>Nitzschia</em> is capable of producing this toxin. Screening for the presence of toxic <em>Nitzschia</em> spp. was carried out at various estuaries in Malaysia. <em>Nitzschia</em>-like cells were isolated and established into clonal cultures. Late stationary phase of cultures were harvested and tested for toxin production using HPLC. Toxin production and compound was verified by LC-MS. From the analyses, at least three cultures were detected with DA, while the rest of the cultures did not show detectable amounts of DA. The localities of the toxic species are Johor and Sabah. Here we conclude that toxic <em>Nitzschia</em> species are present in Malaysian water.</p><p>Keywords: diatom, estuary, intoxication, safety, toxic </p>

Peptidoglycan Recycling in Gram-Positive Bacteria Is Crucial for Survival in Stationary Phase

ABSTRACTPeptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms,Staphylococcus aureus,Bacillus subtilis, andStreptomyces coelicolor, all recycle the sugarN-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ inE. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P toN-acetylglucosamine-6-phosphate andd-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQcells ofS. aureusandB. subtilisrevealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQcultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQmutants ofS. aureusandB. subtilisshowed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival.IMPORTANCEThe peptidoglycan of the bacterial cell wall is turned over steadily during growth. As peptidoglycan fragments were found in large amounts in spent medium of exponentially growing Gram-positive bacteria, their ability to recycle these fragments has been questioned. We conclusively showed recycling of the peptidoglycan component MurNAc in different Gram-positive model organisms and revealed that a MurNAc-6P etherase (MurQ or MurQ ortholog) enzyme is required in this process. We further demonstrated that recycling occurs predominantly during the transition to stationary phase inS. aureusandB. subtilis, explaining why peptidoglycan fragments are found in the medium during exponential growth. We quantified the intracellular accumulation of recycling products in MurNAc-6P etherase gene mutants, revealing that about 5% and 10% of the MurNAc of the cell wall per generation is recycled inS. aureusandB. subtilis, respectively. Importantly, we showed that MurNAc recycling and salvaging does not sustain growth in these bacteria but is used to enhance survival during late stationary phase.

Crystallographic studies of the extracytoplasmic function σ factor σJfromMycobacterium tuberculosis

Mycobacterium tuberculosishas multiple σ factors which enable the bacterium to reprogram its transcriptional machinery under diverse environmental conditions. σJ, an extracytoplasmic function σ factor, is upregulated in late stationary phase cultures and during human macrophage infection. σJgoverns the cellular response to hydrogen peroxide-mediated oxidative stress. σJdiffers from other canonical σ factors owing to the presence of a SnoaL_2 domain at the C-terminus. σJcrystals belonged to the tetragonal space groupI422, with unit-cell parametersa=b= 133.85,c= 75.08 Å. Diffraction data were collected to 2.16 Å resolution on the BM14 beamline at the European Synchrotron Radiation Facility (ESRF).

Coaggregation between Rhodococcus and Acinetobacter strains isolated from the food industry

In this study, coaggregation interactions between Rhodococcus and Acinetobacter strains isolated from food-processing surfaces were characterized. Rhodococcus sp. strain MF3727 formed intrageneric coaggregates with Rhodococcus sp. strain MF3803 and intergeneric coaggregates with 2 strains of Acinetobacter calcoaceticus (MF3293, MF3627). Stronger coaggregation between A. calcoaceticus MF3727 and Rhodococcus sp. MF3293 was observed after growth in batch culture at 30 °C than at 20 °C, after growth in tryptic soy broth than in liquid R2A medium, and between cells in exponential and early stationary phases than cells in late stationary phase. The coaggregation ability of Rhodococcus sp. MF3727 was maintained even after heat and Proteinase K treatment, suggesting its ability to coaggregate was protein independent whereas the coaggregation determinants of the other strains involved proteinaceous cell-surface-associated polymers. Coaggregation was stable at pH 5–9. The mechanisms of coaggregation among Acinetobacter and Rhodococcus strains bare similarity to those displayed by coaggregating bacteria of oral and freshwater origin, with respect to binding between proteinaceous and nonproteinaceous determinants and the effect of environmental factors on coaggregation. Coaggregation may contribute to biofilm formation on industrial food surfaces, protecting bacteria against cleaning and disinfection.

SlyD-dependent nickel delivery limits maturation of [NiFe]-hydrogenases in late-stationary phase Escherichia coli cells

The metallochaperone SlyD is essential for nickel delivery to hydrogenase in stationary phaseE. colicells.