FIG 2 - available via license: CC BY
Content may be subject to copyright.
Analysis of the dual functions of btuD. (A) Vitamin B 12 content of the wild-type (WT) strain, btuD deletion mutant, and btuD complementary strain. (B) The growth speed of the wild-type strain and btuD mutants under different vitamin B 12 conditions (in the presence or absence of 0.5 mM indole). Indole and vitamin B 12 were added to the medium at the beginning of cultivation. (C and D) The survival states of the wild-type strain and btuD mutants under 50 g/ml kanamycin (in the presence or absence of 0.5 mM indole). (E and F) Fluorescence imaging assay of kanamycin transport by the wild-type strain and ΔbtuD mutant under different cultivation conditions. The concentration of indole was 0.5 mM. Indole and fluorescent kanamycin were added 5 h before imaging. The results shown are representative of biological duplicates. The error bars represent the standard deviations for three replicates. For statistical analysis, ***, **, and * indicate P 0.001, P 0.01, and P 0.05, respectively.
Source publication
Bacterial antibiotic resistance modulation by small signaling molecules is an emerging mechanism that has been increasingly reported in recent years. Several studies indicate that indole, an interkingdom signaling molecule, increases bacterial antibiotic resistance. However, the mechanism through which indole reduces antibiotic resistance is largel...
Contexts in source publication
Context 1
... hypothetical proteins. BtuD possesses a conserved P loop/Walker A, Walker B, ABC signature domain, and a Switch domain (31). The amino acid sequence of BtuD showed less than 35% identity to any known ABC transporter ATP-binding protein, and the best hit was BtuD from Salmonella enterica subsp. enterica serovar Typhimurium (identity of 32.0%) (Fig. ...
Context 2
... biochemical assays. We first tested vitamin B 12 uptake in L. enzymogenes YC36 cells with or without exogenous indole. Vitamin B 12 content was determined by enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). We found that 0.5 mM indole significantly improved the absorption efficiency of vitamin B 12 ( Fig. 2A and Fig. S3). While the ΔbtuD mutant had weak vitamin B 12 absorption efficiency, the efficiency of vitamin B 12 uptake was restored in the btuD complementary ΔbtuD::btuD strain ( Fig. 2A), which demonstrated that the uptake of vitamin B 12 was related to the ButD-associated importer. The addition of indole promoted bacterial growth by ...
Context 3
... assay (ELISA) and high-performance liquid chromatography (HPLC). We found that 0.5 mM indole significantly improved the absorption efficiency of vitamin B 12 ( Fig. 2A and Fig. S3). While the ΔbtuD mutant had weak vitamin B 12 absorption efficiency, the efficiency of vitamin B 12 uptake was restored in the btuD complementary ΔbtuD::btuD strain ( Fig. 2A), which demonstrated that the uptake of vitamin B 12 was related to the ButD-associated importer. The addition of indole promoted bacterial growth by increasing the efficiency of vitamin B 12 uptake (Fig. 2B). The ΔbtuD mutant exhibited very slow growth under vitamin B 12 -deficient conditions. Sequence analysis indicated that residues ...
Context 4
... weak vitamin B 12 absorption efficiency, the efficiency of vitamin B 12 uptake was restored in the btuD complementary ΔbtuD::btuD strain ( Fig. 2A), which demonstrated that the uptake of vitamin B 12 was related to the ButD-associated importer. The addition of indole promoted bacterial growth by increasing the efficiency of vitamin B 12 uptake (Fig. 2B). The ΔbtuD mutant exhibited very slow growth under vitamin B 12 -deficient conditions. Sequence analysis indicated that residues Gly48 and Lys49 presumably make extensive hydrogen bonding contacts with the phosphate groups of ADP in the P-loop domain (Fig. S4). Next, we induced point mutations in these residues. The mutant with ...
Context 5
... substitution showed slow growth, especially under vitamin B 12 -deficient conditions. The mutant with K49D single-site substitution possessed decreased vitamin B 12 uptake ability and showed slower growth compared to that of wild-type cells. The growth of the G48Y substitution mutant was unaffected by the tested range of vitamin B 12 conditions (Fig. 2B). Interestingly, the ΔbtuD strain and the mutant with G48Y/K49D double-site substitution did not show IRAR, while IRAR was observed in G48Y and K49D single-site substitution mutants ( Fig. 2C and D). Therefore, we propose that BtuD is a dual-function importer that can transfer both vitamin B 12 and antibiotics. Indole stimulated BtuD ...
Context 6
... slower growth compared to that of wild-type cells. The growth of the G48Y substitution mutant was unaffected by the tested range of vitamin B 12 conditions (Fig. 2B). Interestingly, the ΔbtuD strain and the mutant with G48Y/K49D double-site substitution did not show IRAR, while IRAR was observed in G48Y and K49D single-site substitution mutants ( Fig. 2C and D). Therefore, we propose that BtuD is a dual-function importer that can transfer both vitamin B 12 and antibiotics. Indole stimulated BtuD overexpression and promoted efficient absorption of external vitamin B 12 ; meanwhile, the weak selectivity of the importer caused cells to take up high doses of antibiotics that resulted in cell ...
Context 7
... ester) to kanamycin to produce a fluorescent antibiotic construct, Kana-CFDA (Fig. S6A to C). Dynamic imaging of Kana-CFDA-SE showed that antibiotics accumulated in indole-treated cells, but the entry of antibiotics into the ΔbtuD mutant cells was inhibited, which confirmed that BtuD was responsible for cellular uptake of antibiotics ( Fig. 2E and F). IRAR is common across multiple bacterial species. In subsequent experiments, we found that IRAR is not limited to Lysobacter spp. and is shown by several bacterial species. Pseudoalteromonas is a common pathogenic bacteria and natural product producer that is intrinsically resistant to multiple antibiotics. Our experiments showed that ...
Citations
... In contrast, IAA promoted metabolism in S. aureus to reduce antibiotic tolerance (79). Indole was identified as a signal molecule that either increased and decreased antibiotic resistance by modu lating the expression of transporter and stress resistance genes, as well as by the formation of persister cells (103,104). Although further research is needed to identify the mechanisms of IAA-mediated antibiotic tolerance, our findings support an interplay between compound efflux, membrane permeability, enzymatic antibiotic inactivation and a decrease in bacterial metabolism. ...
The communication between plants and their microbiota is highly dynamic and involves a complex network of signal molecules. Among them, the auxin indole-3-acetic acid (IAA) is a critical phytohormone that not only regulates plant growth and development, but is emerging as an important inter- and intra-kingdom signal that modulates many bacterial processes that are important during interaction with their plant hosts. However, the corresponding signaling cascades remain largely unknown. Here, we advance our understanding of the largely unknown mechanisms by which IAA carries out its regulatory functions in plant-associated bacteria. We showed that IAA caused important changes in the global transcriptome of the rhizobacterium Serratia plymuthica and multidisciplinary approaches revealed that IAA sensing interferes with the signaling mediated by other pivotal plant-derived signals such as amino acids and 4-hydroxybenzoic acid. Exposure to IAA caused large alterations in the transcript levels of genes involved in amino acid metabolism, resulting in significant metabolic alterations. IAA treatment also increased resistance to toxic aromatic compounds through the induction of the AaeXAB pump, which also confers resistance to IAA. Furthermore, IAA promoted motility and severely inhibited biofilm formation; phenotypes that were associated with decreased c-di-GMP levels and capsule production. IAA increased capsule gene expression and enhanced bacterial sensitivity to a capsule-dependent phage. Additionally, IAA induced the expression of several genes involved in antibiotic resistance and led to changes in the susceptibility and responses to antibiotics with different mechanisms of action. Collectively, our study illustrates the complexity of IAA-mediated signaling in plant-associated bacteria.
IMPORTANCE
Signal sensing plays an important role in bacterial adaptation to ecological niches and hosts. This communication appears to be particularly important in plant-associated bacteria since they possess a large number of signal transduction systems that respond to a wide diversity of chemical, physical, and biological stimuli. IAA is emerging as a key inter- and intra-kingdom signal molecule that regulates a variety of bacterial processes. However, despite the extensive knowledge of the IAA-mediated regulatory mechanisms in plants, IAA signaling in bacteria remains largely unknown. Here, we provide insight into the diversity of mechanisms by which IAA regulates primary and secondary metabolism, biofilm formation, motility, antibiotic susceptibility, and phage sensitivity in a biocontrol rhizobacterium. This work has important implications for our understanding of bacterial ecology in plant environments and for the biotechnological and clinical applications of IAA, as well as related molecules.
... Indole, as a signal molecule, has been shown to stimulate persister formation [40,120]. However, the opposite effect of indole on persister frequencies was reported by other studies [121,122]. It was also shown that in M. tuberculosis, the formation of persister cells is associated with a TreS-dependent trehalose-catalytic shift. ...
Trehalose is a naturally occurring, non-reducing saccharide widely distributed in nature. Over the years, research on trehalose has revealed that this initially thought simple storage molecule is a multifunctional and multitasking compound protecting cells against various stress factors. This review presents data on the role of trehalose in maintaining cellular homeostasis under stress conditions and in the virulence of bacteria and fungi. Numerous studies have demonstrated that trehalose acts in the cell as an osmoprotectant, chemical chaperone, free radical scavenger, carbon source, virulence factor, and metabolic regulator. The increasingly researched medical and therapeutic applications of trehalose are also discussed.
... This approach allows for the exploration of sample conformation or the monitoring of interactions. As a demonstration, gram-positive bacteria RT27-A17 (Alkalihalobacillus hwajinpoensis) [54,55] were cultured in 10% Tryptic Soy Broth (TSB) solution, then centrifuged and diluted with deionized water. As shown in Fig. 3, the bacteria were initially captured by a single focused Gaussian beam, and subsequently, the frequency modulation function was enabled on the host. ...
The development of optical tweezers aims to extend their operating function and pattern. However, excessive programming can lead to a decrease in the system’s operating speed and introduce bugs or data transmission delays. In this study, we present a time-shared optical tweezers system that allows for parallel operation of multiple functions. To enable efficient data transmission, we employ a queue structure and a buffer. To assess the system’s performance, we utilize a biological sample in conjunction with the optical tweezers system and scanning imaging technique. We quantify the trapping parameter while concurrently running power stabilization programs. As a result, the standard deviation of the measured stiffness is reduced by 60% in the $x$ x and $y$ y directions and 30% in the $z$ z direction, indicating a significant improvement in calibration precision. Throughout the program execution, the system maintains an operating rate of 110 kHz, and the data are continuously updated in real time on the host. The system’s performance demonstrates its potential for quantification and morphological reconstruction of biological samples.
... Indole, indole acetic acid, polyamines, ammonia, cyclic diguanylate (c-di-GMP), cAMP, 13-methyltetradecanoic acid, and the Pseudomonas quinolone signal are examples of metabolic byproducts that can act as information chemicals and modulate bacterial responses to antibiotics, altering intrinsic resistance to antibiotics and its spread among bacterial cell populations. Additional research into these metabolic byproducts can help identify new antibiotic targets, particularly the important molecular cues that are influencing the rise in intrinsic antibiotic resistance [28][29][30]. One health-based approaches are essential to combating antibiotic resistance because they take into account antibiotics used in both human and veterinary medicine, livestock growth promoters that spread highly mobile ARG across the environment, clinical and animal-associated bacteria, and microbial ecology, such as phage-mediated ARG transfer. ...
Infectious disease mortality have occurred often, raising public awareness of their hazards and advancing global antibiotic research. On the other hand, higher antibiotic levels in natural ecosystems are a result of increased antibiotic demand and use. Gene mutations causing antibiotic resistance in bacterial populations were brought on by natural selection and adaption. The emergence of antibiotic-resistant bacteria was caused by the spread of antibiotic resistance genes in ecosystems, which resulted in a number of antibiotic-resistant diseases on a global scale. An overview of the history of antibiotic discovery, antibiotic use, and antibiotic resistance mechanisms is given in this review study. Bioinformatics is also used to combat antibiotic resistance. To control bacteria that no longer respond to the therapy, the creation of new medications appears to be the primary requirement today.
... Previous studies revealed that L. enzymogenes exhibited high level of multidrug resistance (MDR) to 12 common laboratory antibiotics (Yu and Zhao 2019, 2022. The inter-kingdom signal indole could reverse this high level of resistance by activating a dual importer that could transfer both antibiotics and B 12 into bacterial cells (Wang et al. 2019). In addition, enzymatic modification of chloramphenicol was reported to confer the corresponding resistance via Clp-GNAT (Clp, a global regulator; GNAT, Gcn5related N-acetyltransferase) regulatory system in L. enzymogenes (Zhang et al. 2017). ...
Aims:
The post-transcriptional regulator CsrA regulates many cellular processes, including stress responses in diverse bacteria. However, the role of CsrA in multidrug resistance and biocontrol activity in Lysobacter enzymogenes strain C3 (LeC3) remains unknown.
Methods and results:
In this study, we demonstrated that deletion of the csrA gene resulted in initial slow growth of LeC3 and reduced its resistance to multiple antibiotics, including nalidixic acid (NAL), rifampicin (RIF), kanamycin (Km), and nitrofurantoin (NIT). Loss of the csrA gene also reduced its ability in inhibiting hypha growth of Sclerotium sclerotiorum and influenced its extracellular cellulase and protease activities. Two putative small non-coding regulatory RNAs (sRNAs), referred to as csrB and csrC, were also revealed in the genome of LeC3. Double deletion of csrB and csrC in LeC3 led to increased resistance to NAL, RIF, Km and NIT. However, no difference was observed between LeC3 and the csrB/csrC double mutant in their suppression of S. sclerotiorum hypha growth and production of extracellular enzymes.
Conclusion:
These results suggest that CsrA in LeC3 not only conferred its intrinsic multidrug resistance, but also contributed to its biocontrol activity.
... This was thought to be due to the hyperproduction of K1 β-lactamase since the ESBL/AmpC differential disk results were negative and the GU175 strain was K. oxytoca. If an indole-negative, K1-hyperproducing Indole, a signaling molecule, regulates diverse cellular functions of bacteria, including drug resistance, drug tolerance, biofilm formation, and virulence (6)(7)(8). In this study, a point mutation was found in the SD sequence upstream of tnaC in the indolenegative K. oxytoca GU175 strain. ...
Klebsiella oxytoca is an opportunistic pathogen that causes nosocomial infections. We describe an unusual clinical strain of indole-negative K. oxytoca, GU175, isolated from the urine of a patient with cystitis. The GU175 strain was identified as K. pneumoniae with a probability of 99.40%, negative for indole production, and resistant to third-generation cephalosporins by using the MicroScan Walkaway 40 SI system with the Negative combo EN1 J panel. Biochemical characterization of this strain using a lysine-indole motility medium was negative for indole production. However, identification tests using the MALDI Biotyper system and 16S rRNA gene sequence analysis revealed that GU175 was K. oxytoca. DNA sequence analysis of the tryptophanase operon comparing the GU175 strain with the revertant GU176 strain that tested positive for indole revealed a point mutation in the Shine-Dalgarno sequence upstream of tnaC in the GU175 strain. This is the first report of indole-negative K. oxytoca— attributed to a mutation in the DNA sequence of the tryptophanase operon— isolated from a patient with a urinary tract infection. Since indole-negative K. oxytoca can be misidentified as K. pneumoniae by biochemical characterization, clinical microbiologists should be aware of such misidentifications.
... Indole is an intra-species, interspecies or even interkingdom signaling molecule and plays important biological functions in bacteria and animals (Bansal et al., 2010;Lee & Lee, 2010;Song & Wood, 2020). Cumulative evidence suggests that indole is able to reduce the antibiotic tolerance of Escherichia coli (Hu et al., 2015;Kwan et al., 2015) and Lysobacter enzymogenes (Han et al., 2017;Wang et al., 2019) can be dramatically lowered by indole. In addition, indole also reduces E. coli biofilm formation (Domka, Lee & Wood, 2006;Domka et al., 2007;Lee, Jayaraman & Wood, 2007). ...
... Such suppression effect might be achieved by elevating the expression of efflux pumps that could transport the intracellular antibiotics out of the bacterial cells (Hirakawa et al., 2005) and/or activating stress response-related pathways that could counteract the lethality of the antibiotics (Vega et al., 2012). Nevertheless, several studies revealed that indole can reduce the antibiotic tolerance of E. coli (Hu et al., 2015;Kwan et al., 2015) and Lysobacter enzymogenes (Han et al., 2017;Wang et al., 2019), likely by activating or upregulating antibiotic importers. These contradictory observations might result from the difference in cell growth/metabolic status, as we observed that 5methylindole potentiates aminoglycoside antibiotics under hypoionic conditions against S. aureus cells in stationary-phase but suppress the antibiotics against the cells in exponential-phase (Sun et al., 2020). ...
Antibiotic resistance of bacterial pathogens has become a severe threat to human health. To counteract antibiotic resistance, it is of significance to discover new antibiotics and also improve the efficacy of existing antibiotics. Here we show that 5-methylindole, a derivative of the interspecies signaling molecule indole, is able to directly kill various Gram-positive pathogens ( e.g ., Staphylococcus aureus and Enterococcus faecalis ) and also Gram-negative ones ( e.g ., Escherichia coli and Pseudomonas aeruginosa ), with 2-methylindole being less potent. Particularly, 5-methylindole can kill methicillin-resistant S. aureus , multidrug-resistant Klebsiella pneumoniae , Mycobacterium tuberculosis , and antibiotic-tolerant S. aureus persisters. Furthermore, 5-methylindole significantly potentiates aminoglycoside antibiotics, but not fluoroquinolones, killing of S. aureus . In addition, 5-iodoindole also potentiates aminoglycosides. Our findings open a new avenue to develop indole derivatives like 5-methylindole as antibacterial agents or adjuvants of aminoglycoside.
... sources, mediators of host immune response, and a conception paradigm shift of antibiotics, can nourish and promote the design and implementation of integrative antimicrobial stewardship programs (ASPs) (Yap, 2013). Metabolic byproducts, such as indole (Lee et al., 2010;Wang et al., 2019), indole acetic acid, polyamines, ammonia, cyclic diguanylate (c-di-GMP) (Wang et al., 2011), cAMP (Kwan et al., 2015), 13-methyltetradecanoic acid, and the Pseudomonas quinolone signal, secreted from bacterial cells or present in the bacterial cell milieu, can be infochemicals and modulate bacterial responses toward antibiotics, changing intrinsic resistance to antibiotics and its spread among bacterial cell populations. Further investigation of these metabolic byproducts can inform novel antibiotic targets, especially identification of the key chemical signals mediating increased intrinsic antibiotic resistance. ...
... In Lysobacter Frontiers in Microbiology 03 frontiersin.org enzymogenes, the most studied Lysobacter species; the interkingdom signal indole decreases its resistance against ampicillin and kanamycin by activating a dual importer that could transfer both vitamin B 12 and antibiotics into bacterial cells (Wang et al., 2019). Moreover, cell permeability, β -lactamase activity, and transport were involved in the high level of resistance to ampicillin in L. enzymogenes . ...
Resistance to spectinomycin emerged after widely used for treatment of gonorrhea. Previous studies revealed that Lysobacter enzymogenes strain C3 (LeC3) exhibited elevated level of intrinsic resistance to spectinomycin. In this study, we screened a Tn5 transposon mutant library of LeC3 to elucidate the underlying molecular mechanisms of spectinomycin resistance. Insertion sites in 15 out of 19 mutants recovered with decreased spectinomycin resistance were located on two ribosomal RNA operons at different loci, indicating the pivotal role of ribosomal RNAs in conferring spectinomycin resistance in L. enzymogenes. The other mutants harbored mutations in the tuf, rpoD, mltB, and purB genes. Among them, the tuf and rpoD genes, respectively, encode a translation elongation factor Tu and an RNA polymerase primary sigma factor. They both contribute to protein biosynthesis, where ribosomal RNAs play essential roles. The mltB gene, whose product is involved in cell-wall recycling, was not only associated with resistance against spectinomycin, but also conferred resistance to osmotic stress and ampicillin. In addition, mutation of the purB gene, for which its product is involved in the biosynthesis of inosine and adenosine monophosphates, led to decreased spectinomycin resistance. Addition of exogenous adenine at lower concentration in medium restored the growth deficiency in the purB mutant and increased bacterial resistance to spectinomycin. These results suggest that while cell-wall recycling and purine biosynthesis might contribute to spectinomycin resistance, target rRNAs play critical role in spectinomycin resistance in L. enzymogenes.
... This result indicates that indole enhances the sensitivity of L. enzymogenes to antibiotics through the TCS Le-qseC/ Le-qseB (Han et al. 2017). Recently, Wang et al. (2019c) indicated that exogenous indole can inhibit antibiotic resistance by regulating the expression of a novel importer. In L. enzymogenes, exogenous indole is recognized and transported into cells by the Le-QseC protein and then binds to the Le-QseB protein to regulate the expression of the BtuD importer. ...
... High expression of BtuD importer increases the antibiotic transport efficiency and changes the sensitivity to antibiotics. When indole and antibiotics are used together, a small amount of antibiotics can achieve bactericidal effects, dramatically reducing the dosage of antibiotics and delaying the development of antibiotic resistance (Wang et al. 2019c). ...
... Combined applications with signaling molecules to achieve bactericidal effects have also been studied with other antimicrobial drugs, in addition to antibiotics. The study of Wang et al. (2019c) found that antimicrobial peptides combined with signaling molecules could significantly enhance the bactericidal effect. LED209, a QseC/B inhibitor, is a competitive signaling molecule of indole and can inactivate the QseC/B TCS but has no germicidal effect (Han et al. 2017). ...
Excessive use of antibiotics poses a threat to public health and the environment. In ecosystems, such as the marine environment, antibiotic contamination has led to an increase in bacterial resistance. Therefore, the study of bacterial response to antibiotics and the regulation of resistance formation have become an important research field. Traditionally, the processes related to antibiotic responses and resistance regulation have mainly included the activation of efflux pumps, mutation of antibiotic targets, production of biofilms, and production of inactivated or passivation enzymes. In recent years, studies have shown that bacterial signaling networks can affect antibiotic responses and resistance regulation. Signaling systems mostly alter resistance by regulating biofilms, efflux pumps, and mobile genetic elements. Here we provide an overview of how bacterial intraspecific and interspecific signaling networks affect the response to environmental antibiotics. In doing so, this review provides theoretical support for inhibiting bacterial antibiotic resistance and alleviating health and ecological problems caused by antibiotic contamination.
