Bacterial Stress Adaptation Research Articles

Photoaffinity Capture Compounds to Profile the Magic Spot Nucleotide Interactomes.

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non‐Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species.

Photoaffinity Capture Compounds to Profile the Magic Spot Nucleotide Interactomes**

AbstractMagic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non‐Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species.

The Identity of the Constriction Region of the Ribosomal Exit Tunnel Is Important to Maintain Gene Expression in Escherichia coli.

ABSTRACTMutational changes in bacterial ribosomes often affect gene expression and consequently cellular fitness. Understanding how mutant ribosomes disrupt global gene expression is critical to determining key genetic factors that affect bacterial survival. Here, we describe gene expression and phenotypic changes presented in Escherichia coli cells carrying an uL22(K90D) mutant ribosomal protein, which displayed alterations during growth. Ribosome profiling analyses revealed reduced expression of operons involved in catabolism, indole production, and lysine-dependent acid resistance. In general, translation initiation of proximal genes in several of these affected operons was substantially reduced. These reductions in expression were accompanied by increases in the expression of acid-induced membrane proteins and chaperones, the glutamate-decarboxylase regulon, and the autoinducer-2 metabolic regulon. In agreement with these changes, uL22(K90D) mutant cells had higher glutamate decarboxylase activity, survived better in extremely acidic conditions, and generated more biofilm in static cultures compared to their parental strain. Our work demonstrates that a single mutation in a non-conserved residue of a ribosomal protein affects a substantial number of genes to alter pH resistance and the formation of biofilms.IMPORTANCE All newly synthesized proteins must pass through a channel in the ribosome named the exit tunnel before emerging into the cytoplasm, membrane, and other compartments. The structural characteristics of the tunnel could govern protein folding and gene expression in a species-specific manner but how the identity of tunnel elements influences gene expression is less well-understood. Our global transcriptomics and translatome profiling demonstrate that a single substitution in a non-conserved amino acid of the E. coli tunnel protein uL22 has a profound impact on catabolism, cellular signaling, and acid resistance systems. Consequently, cells bearing the uL22 mutant ribosomes had an increased ability to survive acidic conditions and form biofilms. This work reveals a previously unrecognized link between tunnel identity and bacterial stress adaptation involving pH response and biofilm formation.

The Phosphoarginine Phosphatase PtpB from Staphylococcus aureus Is Involved in Bacterial Stress Adaptation during Infection

Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 ΔptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection.

Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA.

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands – such as “starved” ribosomal complexes, deacylated tRNA and pppGpp – and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native RelA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

Inorganic polyphosphate accumulation suppresses the dormancy response and virulence in Mycobacterium tuberculosis

Stringent response pathways involving inorganic polyphosphate (PolyP) play an essential role in bacterial stress adaptation and virulence. The intracellular levels of PolyP are modulated by the activities of polyphosphate kinase-1 (PPK1), polyphosphate kinase-2 (PPK2), and exopolyphosphatases (PPXs). The genome of Mycobacterium tuberculosis encodes two functional PPXs, and simultaneous deletion of ppx1 and ppx2 results in a defect in biofilm formation. We demonstrate here that these PPXs cumulatively contribute to the ability of M. tuberculosis to survive in nutrient-limiting, low-oxygen growth conditions and also in macrophages. Characterization of single (Δppx2) and double knockout (dkppx) strains of M. tuberculosis indicated that PPX-mediated PolyP degradation is essential for establishing bacterial infection in guinea pigs. RNA-Seq-based transcriptional profiling revealed that relative to the parental strain, the expression levels of DosR regulon-regulated dormancy genes were significantly reduced in the dkppx mutant strain. In concordance, we also provide evidence that PolyP inhibits the autophosphorylation activities associated with DosT and DosS sensor kinases. The results in this study uncover that enzymes involved in PolyP homeostasis play a critical role in M. tuberculosis physiology and virulence and are attractive targets for developing more effective therapeutic interventions.

Optimal Allocation of Bacterial Protein Resources under Nonlethal Protein Maturation Stress

Under different environmental stresses, bacteria optimize the allocation of cellular resources through a variety of mechanisms. Recently, researchers have used phenomenological models to quantitatively characterize the allocation of bacterial protein resources under metabolic and translational limitations. Some stresses interfere with protein maturation, thereby enhancing the expression of chaperones and proteases. However, the reallocation of protein resources caused by such environmental stresses has not been modeled quantitatively. Here, we developed a dynamic model of coarse-grained protein resource fluxes based on a self-replicator that includes protein maturation and degradation. Through flux balance analysis, it produces a constrained optimization problem that can be solved analytically. Accordingly, we predicted protein allocation fractions as functions of growth rate under different limitations, which are basically in line with empirical data. We cultured Escherichia coli in media containing different concentrations of chloramphenicol, acetic acid, and paraquat and measured the functional relationship between the expression level of β-galactosidase driven by a constitutive promoter and the bacterial growth rate, respectively. Taking into account the possible mode of stress limitation on the fluxes, our model reproduces this experimentally measured relationship. In addition, our model is in good agreement with the experimental relationship between growth rate and proteome fraction of unnecessary protein in E. coli, considering the unoptimized upregulation of chaperones with useless protein overexpression. The results provide a more systematic view of bacterial stress adaptation that may help in designing for bioengineering or medical interventions.

Impact of Membrane Phospholipid Alterations in Escherichia coli on Cellular Function and Bacterial Stress Adaptation.

Bacteria have evolved multiple strategies to sense and rapidly adapt to challenging and ever-changing environmental conditions. The ability to alter membrane lipid composition, a key component of the cellular envelope, is crucial for bacterial survival and adaptation in response to environmental stress. However, the precise roles played by membrane phospholipids in bacterial physiology and stress adaptation are not fully elucidated. The goal of this study was to define the role of membrane phospholipids in adaptation to stress and maintenance of bacterial cell fitness. By using genetically modified strains in which the membrane phospholipid composition can be systematically manipulated, we show that alterations in major Escherichia coli phospholipids transform these cells globally. We found that alterations in phospholipids impair the cellular envelope structure and function, the ability to form biofilms, and bacterial fitness and cause phospholipid-dependent susceptibility to environmental stresses. This study provides an unprecedented view of the structural, signaling, and metabolic pathways in which bacterial phospholipids participate, allowing the design of new approaches in the investigation of lipid-dependent processes involved in bacterial physiology and adaptation.IMPORTANCE In order to cope with and adapt to a wide range of environmental conditions, bacteria have to sense and quickly respond to fluctuating conditions. In this study, we investigated the effects of systematic and controlled alterations in bacterial phospholipids on cell shape, physiology, and stress adaptation. We provide new evidence that alterations of specific phospholipids in Escherichia coli have detrimental effects on cellular shape, envelope integrity, and cell physiology that impair biofilm formation, cellular envelope remodeling, and adaptability to environmental stresses. These findings hold promise for future antibacterial therapies that target bacterial lipid biosynthesis.

Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases.

RNA cleavage by bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacterium Deinococcus radiodurans is characterized by highly efficient intrinsic RNA cleavage in comparison with Escherichia coli RNAP. We find that the enhanced RNA cleavage activity largely derives from amino acid substitutions in the trigger loop (TL), a mobile element of the active site involved in various RNAP activities. The differences in RNA cleavage between these RNAPs disappear when the TL is deleted, or in the presence of GreA cleavage factors, which replace the TL in the active site. We propose that the TL substitutions modulate the RNA cleavage activity by altering the TL folding and its contacts with substrate RNA and that the resulting differences in transcriptional proofreading may play a role in bacterial stress adaptation.

The rgg 0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilusLMG18311 thermal adaptation

BackgroundStreptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation.ResultsIn this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311.ConclusionsThese data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

Rhizobium leguminosarum bv. trifolii rosR is required for interaction with clover, biofilm formation and adaptation to the environment

BackgroundRhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.ResultsIn this work, we extend our earlier studies to the characterization of rosR mutants which exhibit pleiotropic phenotypes. The mutants produce three times less exopolysaccharide than the wild type, and the low-molecular-weight fraction in that polymer is greatly reduced. Mutation in rosR also results in quantitative alterations in the polysaccharide constituent of lipopolysaccharide. The rosR mutants are more sensitive to surface-active detergents, antibiotics of the beta-lactam group and some osmolytes, indicating changes in the bacterial membranes. In addition, the rosR mutants exhibit significant decrease in motility and form a biofilm on plastic surfaces, which differs significantly in depth, architecture, and bacterial viability from that of the wild type. The most striking effect of rosR mutation is the considerably decreased attachment and colonization of root hairs, indicating that the mutation affects the first stage of the invasion process. Infection threads initiate at a drastically reduced rate and frequently abort before they reach the base of root hairs. Although these mutants form nodules on clover, they are unable to fix nitrogen and are outcompeted by the wild type in mixed inoculations, demonstrating that functional rosR is important for competitive nodulation.ConclusionsThis report demonstrates the significant role RosR regulatory protein plays in bacterial stress adaptation and in the symbiotic relationship between clover and R. leguminosarum bv. trifolii 24.2.

Microbial inactivation by new technologies of food preservation

The increasing consumer demand for 'fresh-like' foods has led to much research effort in the last 20 years to develop new mild methods for food preservation. Nonthermal methods allow micro-organisms to be inactivated at sublethal temperatures thus better preserving the sensory, nutritional and functional properties of foods. The aim of this review is to provide an overview of the microbiological aspects of the most relevant nonthermal technologies for microbial inactivation currently under study, including irradiation, high hydrostatic pressure, pulsed electric field and ultrasound under pressure. Topics covered are the mechanisms of inactivation, sensitivity of different microbial groups and factors affecting it and kinetics of inactivation.