scholarly journals Isolation of Antibiotic Hypersusceptibility Mutants of Acinetobacter spp. by Selection for DNA Release

2003 ◽  
Vol 47 (4) ◽  
pp. 1267-1274 ◽  
Author(s):  
Hyunwoo Lee ◽  
Nora Vázquez-Laslop ◽  
Katya A. Klyachko ◽  
Alex A. Neyfakh
Keyword(s):  
Bacterial Species ◽  
Marker Gene ◽  
Opportunistic Pathogen ◽  
Bacterial Physiology ◽  
Replica Plating ◽  
Knockout Mutants ◽  
Bacterial Mutants ◽  
Dna Release ◽  
Acinetobacter Spp ◽  
Selection For

ABSTRACT Isolation of bacterial mutants hypersusceptible to antibiotics can reveal novel targets for antibiotic potentiators. However, identification of such mutants is a difficult task which normally requires laborious replica plating of thousands of colonies. The technique proposed here allows for the positive selection of genetic knockout mutants leading to hypersusceptibility. This technique, designated SDR (selection for DNA release), involves introduction of random insertions of a marker gene into the chromosome of a highly transformable bacterial species, followed by treatment of the obtained library with an antibiotic at subinhibitory concentrations. DNA released by lysing bacteria is collected and used to transform fresh bacteria, selecting for insertion of the marker gene. These selection cycles are repeated until variants with a hypersusceptibility phenotype caused by insertion of the marker begin to dominate in the library. This approach allowed for isolation of a number of mutants of the gram-negative opportunistic pathogen Acinetobacter sp. susceptible to 4- to 16-times-lower concentrations of ampicillin than wild-type bacteria. The mutations affected proteins involved in peptidoglycan turnover and, surprisingly, proteins involved in exopolysaccharide production. A further modification of the SDR technique is described which allows for selecting mutants hypersensitive to agents that affect bacterial physiology but do not cause cell lysis, e.g., inhibitors of translation. This application of SDR is illustrated here by identification of several mutants of Acinetobacter sp. with increased susceptibility (two- to fivefold decrease in the MIC) to erythromycin. The same technique can be used to identify prospective targets for potentiators of many other antibacterial agents.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

scholarly journals No Change, No Life? What We Know about Phase Variation in Staphylococcus aureus

2021 ◽  
Vol 9 (2) ◽  
pp. 244
Author(s):  
Vishal Gor ◽  
Ryosuke L. Ohniwa ◽  
Kazuya Morikawa
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
High Frequency ◽  
Direct Evidence ◽  
Bacterial Species ◽  
Phase Variation ◽  
Opportunistic Pathogen ◽  
Adaptation Strategy ◽  
Innate Immune ◽  
Epigenetic Mechanisms

Phase variation (PV) is a well-known phenomenon of high-frequency reversible gene-expression switching. PV arises from genetic and epigenetic mechanisms and confers a range of benefits to bacteria, constituting both an innate immune strategy to infection from bacteriophages as well as an adaptation strategy within an infected host. PV has been well-characterized in numerous bacterial species; however, there is limited direct evidence of PV in the human opportunistic pathogen Staphylococcus aureus. This review provides an overview of the mechanisms that generate PV and focuses on earlier and recent findings of PV in S. aureus, with a brief look at the future of the field.

scholarly journals Bacterial response to graphene oxide and reduced graphene oxide integrated in agar plates

2018 ◽  
Vol 5 (11) ◽  
pp. 181083 ◽  
Author(s):  
V. R. S. S. Mokkapati ◽  
Santosh Pandit ◽  
Jinho Kim ◽  
Anders Martensson ◽  
Martin Lovmar ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Reduced Graphene Oxide ◽  
Bacterial Growth ◽  
Bacterial Species ◽  
Bacterial Activity ◽  
Bacterial Physiology ◽  
Bacterial Response ◽  
Solid Agar ◽  
Reduced Graphene ◽  
Set Up

There are contradictory reports in the literature regarding the anti-bacterial activity of graphene, graphene oxide (GO) and reduced graphene oxide (rGO). This controversy is mostly due to variations in key parameters of the reported experiments, like: type of substrate, form of graphene, number of layers, type of solvent and most importantly, type of bacteria. Here, we present experimental data related to bacterial response to GO and rGO integrated in solid agar-based nutrient plates—a standard set-up for bacterial growth that is widely used by microbiologists. Bacillus subtilis and Pseudomonas aeruginosa strains were used for testing bacterial growth. We observed that plate-integrated rGO showed strong anti-bacterial activity against both bacterial species. By contrast, plate-integrated GO was harmless to both bacteria. These results reinforce the notion that the response of bacteria depends critically on the type of graphene material used and can vary dramatically from one bacterial strain to another, depending on bacterial physiology.

scholarly journals Bifidobacterium adolescentis shows potential to strengthen host defence against gastrointestinal infection via inhibition of the opportunistic pathogen Candida albicans and stimulation of human-isolated macrophages killing capacity in vitro

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Liviana Ricci ◽  
Joanna Mackie ◽  
Megan D. Lenardon ◽  
Caitlin Jukes ◽  
Ahmed N. Hegazy ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Bacterial Species ◽  
Opportunistic Pathogen ◽  
Limited Information ◽  
Human Gut ◽  
Bifidobacterium Adolescentis ◽  
Host Immune Responses ◽  
Opportunistic Fungal Pathogen ◽  
Antimicrobial Metabolites

The human gut microbiota enhances the host’s resistance to enteric pathogens via colonisation resistance, a phenomenon that is driven by multiple mechanisms, such as production of antimicrobial metabolites and activation of host immune responses. However, there is limited information on how individual gut bacterial species, particularly many of the dominant anaerobes, might impact the host’s defence. This study investigated the potential of specific human gut isolates to bolster the host’s resistance to infection. First, by antagonising the opportunistic fungal pathogen Candida albicans, and secondly, by modulating the killing capacity of human-isolated macrophages in vitro. Co-culturing C. albicans with faecal microbiota from different healthy individuals revealed varying levels of fungal inhibition. In vitro assays with a panel of representative human gut anaerobes confirmed that culture supernatants from certain bacterial isolates, in particular of Bifidobacterium adolescentis, significantly inhibited C. albicans growth. Mechanistic studies revealed that microbial fermentation acids including acetate and lactate, in combination with the associated decrease in pH, were strong drivers of this inhibitory activity. In the second in vitro assay, human-isolated macrophages were exposed to bacterial supernatants, and subsequently tested for their capacity to eliminate adherent-invasive Escherichia coli. Among the gut anaerobes tested, B. adolescentis was revealed to exert the strongest immunostimulatory and killing effect when compared to the unstimulated macrophages control. B. adolescentis is known to be stimulated by dietary consumption of resistant starch andmay therefore represent an attractive target for the development of probiotic and prebiotic interventions tailored to enhancethe host’s natural defences against infection.

scholarly journals The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferons by engaging cytosolic DNA sensing in macrophages

2021 ◽  
Author(s):  
Krystal J Vail ◽  
Bibiana Petri da Silveira ◽  
Samantha L Bell ◽  
Angela I Bordin ◽  
Noah D Cohen ◽  
...  
Keyword(s):  
Bacterial Pathogen ◽  
Opportunistic Pathogen ◽  
Rhodococcus Equi ◽  
Type I Interferons ◽  
Type I ◽  
Type I Ifn ◽  
Dna Sensing ◽  
Interferon Stimulated Genes ◽  
Galectin 3 ◽  
Dna Release

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics, demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA surveillance pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this we found that a population of ~12% of R. equi phagosomes recruited the galectin-3, -8 and -9 danger receptors. Interesting, neither phagosomal damage nor induction of type I IFN required the R. equi's virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulated ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.

scholarly journals The functional ClpXP protease of Chlamydia trachomatis requires distinct clpP genes from separate genetic loci

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Stefan Pan ◽  
Imran T. Malik ◽  
Dhana Thomy ◽  
Beate Henrichfreise ◽  
Peter Sass
Keyword(s):  
Chlamydia Trachomatis ◽  
Bacterial Species ◽  
Human Pathogens ◽  
Bacterial Physiology ◽  
Sexually Transmitted ◽  
Clp Proteases ◽  
Obligate Intracellular Pathogen ◽  
Antibacterial Target ◽  
Insight Into

Abstract Clp proteases play a central role in bacterial physiology and, for some bacterial species, are even essential for survival. Also due to their conservation among bacteria including important human pathogens, Clp proteases have recently attracted considerable attention as antibiotic targets. Here, we functionally reconstituted and characterized the ClpXP protease of Chlamydia trachomatis (ctClpXP), an obligate intracellular pathogen and the causative agent of widespread sexually transmitted diseases in humans. Our in vitro data show that ctClpXP is formed by a hetero-tetradecameric proteolytic core, composed of two distinct homologs of ClpP (ctClpP1 and ctClpP2), that associates with the unfoldase ctClpX via ctClpP2 for regulated protein degradation. Antibiotics of the ADEP class interfere with protease functions by both preventing the interaction of ctClpX with ctClpP1P2 and activating the otherwise dormant proteolytic core for unregulated proteolysis. Thus, our results reveal molecular insight into ctClpXP function, validating this protease as an antibacterial target.

scholarly journals Spatiotemporal Distribution of Pseudomonas aeruginosa Alkyl Quinolones under Metabolic and Competitive Stress

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Tianyuan Cao ◽  
Jonathan V. Sweedler ◽  
Paul W. Bohn ◽  
Joshua D. Shrout
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Species ◽  
Metabolic Stress ◽  
Opportunistic Pathogen ◽  
Cell Interaction ◽  
Chemical Imaging ◽  
Carbon Limitation ◽  
Bacterial Strains ◽  
Content Type ◽  
High Level

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen important to diseases such as cystic fibrosis. P. aeruginosa has multiple quorum-sensing (QS) systems, one of which utilizes the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]). Here, we use hyperspectral Raman imaging to elucidate the spatiotemporal PQS distributions that determine how P. aeruginosa regulates surface colonization and its response to both metabolic stress and competition from other bacterial strains. These chemical imaging experiments illustrate the strong link between environmental challenges, such as metabolic stress caused by nutritional limitations or the presence of another bacterial species, and PQS signaling. Metabolic stress elicits a complex response in which limited nutrients induce the bacteria to produce PQS earlier, but the bacteria may also pause PQS production entirely if the nutrient concentration is too low. Separately, coculturing P. aeruginosa in the proximity of another bacterial species, or its culture supernatant, results in earlier production of PQS. However, these differences in PQS appearance are not observed for all alkyl quinolones (AQs) measured; the spatiotemporal response of 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) is highly uniform for most conditions. These insights on the spatiotemporal distributions of quinolones provide additional perspective on the behavior of P. aeruginosa in response to different environmental cues. IMPORTANCE Alkyl quinolones (AQs), including Pseudomonas quinolone signal (PQS), made by the opportunistic pathogen Pseudomonas aeruginosa have been associated with both population density and stress. The regulation of AQ production is known to be complex, and the stimuli that modulate AQ responses are not fully clear. Here, we have used hyperspectral Raman chemical imaging to examine the temporal and spatial profiles of AQs exhibited by P. aeruginosa under several potentially stressful conditions. We found that metabolic stress, effected by carbon limitation, or competition stress, effected by proximity to other species, resulted in accelerated PQS production. This competition effect did not require cell-to-cell interaction, as evidenced by the fact that the addition of supernatants from either Escherichia coli or Staphylococcus aureus led to early appearance of PQS. Lastly, the fact that these modulations were observed for PQS but not for all AQs suggests a high level of complexity in AQ regulation that remains to be discerned.

scholarly journals Regulation of OmpA Translation and Shigella dysenteriae Virulence by an RNA Thermometer

2019 ◽  
Vol 88 (3) ◽  
Author(s):  
Erin R. Murphy ◽  
Johanna Roßmanith ◽  
Jacob Sieg ◽  
Megan E. Fris ◽  
Hebaallaha Hussein ◽  
...  
Keyword(s):  
In Silico ◽  
Bacterial Species ◽  
Regulation Of Gene Expression ◽  
Structure And Function ◽  
Shigella Dysenteriae ◽  
Content Type ◽  
Bacterial Physiology ◽  
Wide Range ◽  
Rna Thermometer ◽  
And Function

ABSTRACT RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae. First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.

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