scholarly journals Pseudomonas syringae pv. phaseolicola Uses Distinct Modes of Stationary-Phase Persistence To Survive Bacteriocin and Streptomycin Treatments

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Ravikumar R. Patel ◽  
Prem P. Kandel ◽  
Eboni Traverso ◽  
Kevin L. Hockett ◽  
Lindsay R. Triplett
Keyword(s):  
Pseudomonas Syringae ◽  
Metabolic Activity ◽  
Recurrent Infection ◽  
Small Population ◽  
Antimicrobial Treatment ◽  
Intact Cells ◽  
Content Type ◽  
Redox States ◽  
Metabolic States ◽  
Metabolic Signal

ABSTRACT Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of their persistence in the presence of membrane-disrupting biological compounds is less well understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here, we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population in the redox and membrane permeability state. Intact cells were sorted into three fractions according to metabolic activity, as indicated by a redox-sensing reporter dye. Streptomycin persisters were cultured from the fraction associated with the lowest metabolic activity, but tailocin persisters were cultured from a fraction associated with an active metabolic signal. Cells from culturable fractions were able to infect host plants, while the nonculturable fractions were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct metabolic states associated with antibiotic persistence, tailocin persistence, and loss of virulence and demonstrates that tailocin is highly effective in eliminating dormant cells. IMPORTANCE Populations of genetically identical bacteria encompass heterogeneous physiological states. The small fraction of bacteria that are dormant can help the population survive exposure to antibiotics and other stresses, potentially contributing to recurring infection cycles in animal or plant hosts. Membrane-disrupting biological control treatments are effective in killing dormant bacteria, but these treatments also leave persister-like survivors. The current work demonstrates that in Pph, persisters surviving treatment with membrane-disrupting tailocin proteins have an elevated redox state compared to that of dormant streptomycin persisters. Combination treatment was effective in killing both persister types. Culturable persisters corresponded closely with infectious cells in each treated population, whereas the high-redox and unculturable fractions were not infectious. In linking redox states to heterogeneous phenotypes of tailocin persistence, streptomycin persistence, and infection capability, this work will inform the search for mechanisms and markers for each phenotype.

scholarly journals A Pseudomonas plant pathogen uses distinct modes of stationary phase persistence to survive bacteriocin and streptomycin treatments

2021 ◽  
Author(s):  
Ravikumar R. Patel ◽  
Prem P. Kandel ◽  
Eboni Traverso ◽  
Kevin L. Hockett ◽  
Lindsay R. Triplett
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Pathogen ◽  
Host Plants ◽  
Recurrent Infection ◽  
Small Population ◽  
Antimicrobial Treatment ◽  
Redox Activity ◽  
Redox States ◽  
Redox Active ◽  
Biological Compounds

Abstract Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of persistence to membrane-disrupting biological compounds is less well-understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin, and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population of redox and membrane permeability states. Sorting cells according to redox reporter intensity identified streptomycin persisters among the low-redox fraction, but tailocin persisters were only cultured from the fraction with intermediate redox activity. Cells from culturable fractions were able to infect host plants, while nonculturable redox-active cells were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct redox states associated with antibiotic persistence, tailocin persistence, and virulence, and demonstrates that tailocin is highly effective in eliminating dormant cells.

scholarly journals Exometabolite Dynamics over Stationary Phase Reveal Strain-Specific Responses

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
pp. e00493-20
Author(s):  
John L. Chodkowski ◽  
Ashley Shade
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Syringae ◽  
Stationary Phases ◽  
Interspecies Interactions ◽  
Bacterial Strains ◽  
Community Interactions ◽  
Succinate Production ◽  
Intact Cells ◽  
Content Type ◽  
Environmental Bacteria

ABSTRACTMicrobial exponential growth is expected to occur infrequently in environments that have long periods of nutrient starvation punctuated by short periods of high nutrient flux. These conditions likely impose nongrowth states for microbes. However, nongrowth states are uncharacterized for the majority of environmental bacteria, especially in regard to exometabolite production. We compared exometabolites produced over stationary phase across three environmental bacteria: Burkholderia thailandensis E264 (ATCC 700388), Chromobacterium violaceum ATCC 31532, and Pseudomonas syringae pv. tomato DC3000 (ATCC BAA-871). We grew each strain in monoculture and investigated exometabolite dynamics from mid-exponential to stationary phases. We focused on exometabolites that were released into the medium and accumulated over 45 h, including approximately 20 h of stationary phase. We also analyzed transcripts (transcriptome sequencing [RNA-seq]) to interpret exometabolite output. We found that the majority of exometabolites released were strain specific, with a subset of identified exometabolites involved in both central and secondary metabolism. Transcript analysis supported that exometabolites were released from intact cells, as various transporters had either increased or consistent transcripts through time. Interestingly, we found that succinate was one of the most abundant identifiable exometabolites for all strains and that each strain rerouted their metabolic pathways involved in succinate production during stationary phase. These results show that nongrowth states can be metabolically dynamic and that environmental bacteria can enrich a minimal environment with diverse chemical compounds as a consequence of growth and postgrowth maintenance in stationary phase. This work provides insights into microbial community interactions via exometabolites under conditions of growth cessation or limitation.IMPORTANCE Nongrowth states are common for bacteria that live in environments that are densely populated and predominantly nutrient exhausted, and yet these states remain largely uncharacterized in cellular metabolism and metabolite output. Here, we investigated and compared stationary-phase exometabolites and RNA transcripts for each of three environmental bacterial strains. We observed that diverse exometabolites were produced and provide evidence that these exometabolites accumulate over time through release by intact cells. Additionally, each bacterial strain had a characteristic exometabolite profile and exhibited dynamics in exometabolite composition. This work affirms that stationary phase is metabolically dynamic, with each strain tested creating a unique chemical signature in the extracellular space and altering metabolism in stationary phase. These findings set the stage for understanding how bacterial populations can support surrounding neighbors in environments with prolonged nutrient exhaustion through exometabolite-mediated interspecies interactions.

scholarly journals In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

2011 ◽  
Vol 56 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Marisa H. Miceli ◽  
Stella M. Bernardo ◽  
T. S. Neil Ku ◽  
Carla Walraven ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
High Dose ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Content Type ◽  
Heparin Sodium ◽  
The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

scholarly journals In VitroActivity of Glucosinolates and Their Degradation Products against Brassica-Pathogenic Bacteria and Fungi

2014 ◽  
Vol 81 (1) ◽  
pp. 432-440 ◽  
Author(s):  
T. Sotelo ◽  
M. Lema ◽  
P. Soengas ◽  
M. E. Cartea ◽  
P. Velasco
Keyword(s):  
Pseudomonas Syringae ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Degradation Products ◽  
Allyl Isothiocyanate ◽  
Leaf Extracts ◽  
Soil Pathogens ◽  
Content Type ◽  
Positive Effects

ABSTRACTGlucosinolates (GSLs) are secondary metabolites found inBrassicavegetables that confer on them resistance against pests and diseases. Both GSLs and glucosinolate hydrolysis products (GHPs) have shown positive effects in reducing soil pathogens. Information about theirin vitrobiocide effects is scarce, but previous studies have shown sinigrin GSLs and their associated allyl isothiocyanate (AITC) to be soil biocides. The objective of this work was to evaluate the biocide effects of 17 GSLs and GHPs and of leaf methanolic extracts of different GSL-enrichedBrassicacrops on suppressingin vitrogrowth of two bacterial (Xanthomonas campestrispv. campestris andPseudomonas syringaepv. maculicola) and two fungal (AlternariabrassicaeandSclerotiniascletoriorum)Brassicapathogens. GSLs, GHPs, and methanolic leaf extracts inhibited the development of the pathogens tested compared to the control, and the effect was dose dependent. Furthermore, the biocide effects of the different compounds studied were dependent on the species and race of the pathogen. These results indicate that GSLs and their GHPs, as well as extracts of differentBrassicaspecies, have potential to inhibit pathogen growth and offer new opportunities to study the use ofBrassicacrops in biofumigation for the control of multiple diseases.

Viability and respiratory activity ofPseudomonas syringaecells starved in buffer

1995 ◽  
Vol 41 (4-5) ◽  
pp. 372-377 ◽  
Author(s):  
João P. S. Cabral
Keyword(s):  
Oxygen Consumption ◽  
Electron Transport ◽  
Pseudomonas Syringae ◽  
Respiratory Activity ◽  
Bacterial Cells ◽  
Intact Cells ◽  
Rate Of Oxygen Consumption ◽  
Metabolic Activities ◽  
Different Levels ◽  
Number Of Cells

Pseudomonas syringae cells starved in buffer released orcinol-reactive molecules and materials that absorbed ultraviolet light. The number of cells culturable in nutrient medium decreased more rapidly than the number of intact particles determined by microscopy. The results suggested that starvation resulted in the lysis of an increasing number of cells, and that a fraction of the intact particles were not culturable. Starvation also resulted in a decrease in the rate of oxygen consumption with acetate, glycerol, and succinate, but at different levels. Whereas the respiration of acetate and glycerol decreased concomitantly with culturability, the respiration of succinate decreased to levels similar to the concentration of intact cells, suggesting that all intact particles respired the succinate, but only the culturable cells respired the acetate and glycerol. The results suggest that measuring the activity of the electron-transport system can overestimate the viability of starved bacterial cells, and that complex metabolic activities such as the respiration of acetate and glycerol are probably better suited for the evaluation of this parameter.Key words: Pseudomonas syringae, starvation, culturability, viability, respiration.

scholarly journals Methanogens and Methanogenesis in the Rumens and Ceca of Lambs Fed Two Different High-Grain-Content Diets

2012 ◽  
Vol 79 (6) ◽  
pp. 1777-1786 ◽  
Author(s):  
M. Popova ◽  
D. P. Morgavi ◽  
C. Martin
Keyword(s):  
Metabolic Activity ◽  
Methane Production ◽  
Direct Evidence ◽  
Species Level ◽  
Ex Situ ◽  
Content Type ◽  
Copy Numbers ◽  
Dietary Starch ◽  
Induced Changes

ABSTRACTThe amount and nature of dietary starch are known to influence the extent and site of feed digestion in ruminants. However, how starch degradability may affect methanogenesis and methanogens along the ruminant's digestive tract is poorly understood. This study examined the diversity and metabolic activity of methanogens in the rumen and cecum of lambs receiving wheat or corn high-grain-content diets. Methane productionin vivoandex situwas also monitored.In vivodaily methane emissions (CH4g/day) were 36% (P< 0.05) lower in corn-fed lambs than in wheat-fed lambs.Ex situmethane production (μmol/h) was 4-fold higher for ruminal contents than for cecal contents (P< 0.01), while methanogens were 10-fold higher in the rumen than in the cecum (mcrAcopy numbers;P< 0.01). Clone library analysis indicated thatMethanobrevibacterwas the dominant genus in both sites. Diet induced changes at the species level, as theMethanobrevibacter millerae-M. gottschalkii-M. smithiiclade represented 78% of the sequences from the rumen of wheat-fed lambs and just about 52% of the sequences from the rumen of the corn-fed lambs. Diet did not affectmcrAexpression in the rumen. In the cecum, however, expression was 4-fold and 2-fold lower than in the rumen for wheat- and corn-fed lambs, respectively. Though we had no direct evidence for compensation of reduced rumen methane production with higher cecum methanogenesis, the ecology of methanogens in the cecum should be better considered.

scholarly journals Complete and Draft Genome Sequences of the Cruciferous Pathogens Pseudomonas cannabina pv. alisalensis and Pseudomonas syringae pv. maculicola

2021 ◽  
Vol 10 (17) ◽  
Author(s):  
Takashi Fujikawa ◽  
Yuichi Takikawa ◽  
Yasuhiro Inoue
Keyword(s):  
Pseudomonas Syringae ◽  
Leaf Spot ◽  
Draft Genome ◽  
Leaf Blight ◽  
Bacterial Leaf Blight ◽  
Genome Sequences ◽  
Bacterial Leaf Spot ◽  
Content Type

ABSTRACT Pseudomonas cannabina pv. alisalensis and Pseudomonas syringae pv. maculicola cause bacterial leaf blight and bacterial leaf spot of crucifers (Brassicaceae). Both pathogens are threats to the cultivation of cruciferous crops. Here, we sequenced two strains of each pathogen, which will contribute to the development of countermeasures for the above diseases.

scholarly journals Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Physical Interaction ◽  
Flagellar Motor ◽  
Intact Cells ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cross Links ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

scholarly journals Evolutionary Plasticity of AmrZ Regulation in Pseudomonas

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. e00132-18 ◽  
Author(s):  
David A. Baltrus ◽  
Kevin Dougherty ◽  
Beatriz Diaz ◽  
Rachel Murillo
Keyword(s):  
Pseudomonas Syringae ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Dual Function ◽  
Genomic Context ◽  
Master Regulator ◽  
Content Type ◽  
Swimming Motility ◽  
Positive Regulator ◽  
Mode Of Regulation

ABSTRACT amrZ encodes a master regulator protein conserved across pseudomonads, which can be either a positive or negative regulator of swimming motility depending on the species examined. To better understand plasticity in the regulatory function of AmrZ, we characterized the mode of regulation for this protein for two different motility-related phenotypes in Pseudomonas stutzeri. As in Pseudomonas syringae, AmrZ functions as a positive regulator of swimming motility within P. stutzeri, which suggests that the functions of this protein with regard to swimming motility have switched at least twice across pseudomonads. Shifts in mode of regulation cannot be explained by changes in AmrZ sequence alone. We further show that AmrZ acts as a positive regulator of colony spreading within this strain and that this regulation is at least partially independent of swimming motility. Closer investigation of mechanistic shifts in dual-function regulators like AmrZ could provide unique insights into how transcriptional pathways are rewired between closely related species. IMPORTANCE Microbes often display finely tuned patterns of gene regulation across different environments, with major regulatory changes controlled by a small group of “master” regulators within each cell. AmrZ is a master regulator of gene expression across pseudomonads and can be either a positive or negative regulator for a variety of pathways depending on the strain and genomic context. Here, we demonstrate that the phenotypic outcomes of regulation of swimming motility by AmrZ have switched at least twice independently in pseudomonads, so that AmrZ promotes increased swimming motility in P. stutzeri and P. syringae but represses this phenotype in Pseudomonas fluorescens and Pseudomonas aeruginosa. Since examples of switches in regulatory mode are relatively rare, further investigation into the mechanisms underlying shifts in regulator function for AmrZ could provide unique insights into the evolution of bacterial regulatory proteins.

scholarly journals Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Eric C. DiBiasio ◽  
Hilary J. Ranson ◽  
James R. Johnson ◽  
David C. Rowley ◽  
Paul S. Cohen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Recurrent Infection ◽  
Colony Growth ◽  
Quiescent State ◽  
Content Type ◽  
Tract Infections

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling. IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

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