scholarly journals Nitric Oxide-Mediated Induction of Dispersal inPseudomonas aeruginosaBiofilms Is Inhibited by Flavohemoglobin Production and Is Enhanced by Imidazole

2017 ◽  
Vol 62 (3) ◽  
Author(s):  
Xinyi Zhu ◽  
Hyun-Suk Oh ◽  
Yu Chiu Beverly Ng ◽  
Pei Yi Peggy Tang ◽  
Nicolas Barraud ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Control Strategies ◽  
Bacterial Species ◽  
Pcr Analysis ◽  
Signal Molecule ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biofilm Dispersal ◽  
New Perspective ◽  
Underlying Mechanisms

ABSTRACTThe biological signal molecule nitric oxide (NO) was found to induce biofilm dispersal across a range of bacterial species, which led to its consideration for therapeutic strategies to treat biofilms and biofilm-related infections. However, biofilms are often not completely dispersed after exposure to NO. To better understand this phenomenon, we investigated the response ofPseudomonas aeruginosabiofilm cells to successive NO treatments. When biofilms were first pretreated with a low, noneffective dose of NO, a second dose of the signal molecule at a concentration usually capable of inducing dispersal did not have any effect. Amperometric analysis revealed that pretreatedP. aeruginosacells had enhanced NO-scavenging activity, and this effect was associated with the production of the flavohemoglobin Fhp. Further, quantitative real-time reverse transcription-PCR (qRT-PCR) analysis showed thatfhpexpression increased by over 100-fold in NO-pretreated biofilms compared to untreated biofilms. Biofilms of mutant strains harboring mutations infhporfhpR, encoding a NO-responsive regulator offhp, were not affected in their dispersal response after the initial pretreatment with NO. Overall, these results suggest that FhpR can sense NO to trigger production of the flavohemoglobin Fhp and inhibit subsequent dispersal responses to NO. Finally, the addition of imidazole, which can inhibit the NO dioxygenase activity of flavohemoglobin, attenuated the prevention of dispersal after NO pretreatment and improved the dispersal response in older, starved biofilms. This study clarifies the underlying mechanisms of impaired dispersal induced by repeated NO treatments and offers a new perspective for improving the use of NO in biofilm control strategies.

scholarly journals Nitric Oxide and Iron Signaling Cues Have Opposing Effects on Biofilm Development inPseudomonas aeruginosa

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Xinyi Zhu ◽  
Scott A. Rice ◽  
Nicolas Barraud
Keyword(s):  
Nitric Oxide ◽  
Cellular Response ◽  
Control Strategies ◽  
Iron Acquisition ◽  
Iron Chelator ◽  
Content Type ◽  
Environmental Signals ◽  
Biofilm Control ◽  
Biofilm Dispersal ◽  
Biofilm Development

ABSTRACTWhile both iron and nitric oxide (NO) are redox-active environmental signals shown to regulate biofilm development, their interaction and roles in regulating biofilms have not been fully elucidated. In this study, exposure ofPseudomonas aeruginosabiofilms to exogenous NO inhibited the expression of iron acquisition-related genes and the production of the siderophore pyoverdine. Furthermore, supplementation of the culture medium with high levels of iron (100 μM) counteracted NO-induced biofilm dispersal by promoting the rapid attachment of planktonic cells. In the presence of iron, biofilms were found to disperse transiently to NO, while the freshly dispersed cells reattached rapidly within 15 min. This effect was not due to the scavenging of NO by free iron but involved a cellular response induced by iron that led to the elevated production of the exopolysaccharide Psl. Interestingly, most Psl remained on the substratum after treatment with NO, suggesting that dispersal involved changes in the interactions between Psl andP. aeruginosacells. Taken together, our results suggest that iron and NO regulate biofilm development via different pathways, both of which include the regulation of Psl-mediated attachment. Moreover, the addition of an iron chelator worked synergistically with NO in the dispersal of biofilms.IMPORTANCENitric oxide (NO), which induces biofilm dispersal, is a promising strategy for biofilm control in both clinical and industrial contexts. However, competing environmental signals may reduce the efficacy of NO. The results presented here suggest that the presence of iron represents one such environmental cue that antagonizes the activity of NO as a biofilm-dispersing agent. Based on this understanding, we developed a strategy to enhance dispersal by combining NO with an iron-scavenging agent. Overall, this study links two important environmental signals, iron and NO, with their roles in biofilm development and suggests new ways for improving the use of NO in biofilm control strategies.

scholarly journals Low Concentrations of Nitric Oxide Modulate Streptococcus pneumoniae Biofilm Metabolism and Antibiotic Tolerance

2016 ◽  
Vol 60 (4) ◽  
pp. 2456-2466 ◽  
Author(s):  
Raymond N. Allan ◽  
Samantha Morgan ◽  
Sanjita Brito-Mutunayagam ◽  
Paul Skipp ◽  
Martin Feelisch ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Streptococcus Pneumoniae ◽  
Ex Vivo ◽  
Bacterial Species ◽  
Antibiotic Tolerance ◽  
Content Type ◽  
Low Concentration ◽  
Low Concentrations ◽  
Biofilm Dispersal ◽  
Novel Therapeutic

ABSTRACTStreptococcus pneumoniaeis one of the key pathogens responsible for otitis media (OM), the most common infection in children and the largest cause of childhood antibiotic prescription. Novel therapeutic strategies that reduce the overall antibiotic consumption due to OM are required because, although widespread pneumococcal conjugate immunization has controlled invasive pneumococcal disease, overall OM incidence has not decreased. Biofilm formation represents an important phenotype contributing to the antibiotic tolerance and persistence ofS. pneumoniaein chronic or recurrent OM. We investigated the treatment of pneumococcal biofilms with nitric oxide (NO), an endogenous signaling molecule and therapeutic agent that has been demonstrated to trigger biofilm dispersal in other bacterial species. We hypothesized that addition of low concentrations of NO to pneumococcal biofilms would improve antibiotic efficacy and that higher concentrations exert direct antibacterial effects. Unlike in many other bacterial species, low concentrations of NO did not result inS. pneumoniaebiofilm dispersal. Instead, treatment of bothin vitrobiofilms andex vivoadenoid tissue samples (a reservoir forS. pneumoniaebiofilms) with low concentrations of NO enhanced pneumococcal killing when combined with amoxicillin-clavulanic acid, an antibiotic commonly used to treat chronic OM. Quantitative proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) identified 13 proteins that were differentially expressed following low-concentration NO treatment, 85% of which function in metabolism or translation. Treatment with low-concentration NO, therefore, appears to modulate pneumococcal metabolism and may represent a novel therapeutic approach to reduce antibiotic tolerance in pneumococcal biofilms.

scholarly journals Identification of Nitric Oxide Responsive Genes in the Rudimentary Leaves of Litchi chinensis

2018 ◽  
Vol 46 (2) ◽  
pp. 608-614
Author(s):  
Xingyu LU ◽  
Houbin CHEN ◽  
Zhiqun HU ◽  
Biyan ZHOU
Keyword(s):  
Nitric Oxide ◽  
Hot Spring ◽  
Subtractive Hybridization ◽  
Hypothetical Protein ◽  
Pcr Analysis ◽  
Litchi Chinensis ◽  
Tropical Regions ◽  
Reverse Transcription Pcr ◽  
Protein Encoding ◽  
Floral Differentiation

Litchi (Litchi chinensis Sonn.) is an evergreen woody fruit tree widely cultivated in subtropical and tropical regions. Warm winter and hot spring often leads to abnormal floral differentiation in litchi. Under this condition, the rudimentary leaves in the floral buds expand and the inflorescences will stop developing. Thus, how to promote abortion of rudimentary leaves in litchi inflorescence are important for floral development. Previous study indicated that nitric oxide (NO) produced by sodium nitroprusside (SNP) promoted flowering and abortion of rudimentary leaves in litchi. In the present study, a suppression subtractive hybridization (SSH) was used to identify NO responsive genes. As a result, 16 high homologous ESTs were obtained from the SSH library of the SNP treated rudimentary leaves. The ESTs were classified into three groups. They are disease/defensive, protein destination and storage, and protein synthesis. Quantitative reverse transcription PCR (qRT-PCR) analysis indicated that 6 out of the 7 randomly selected ESTs’expression showed an increasing trend from 0 h to 10 h of SNP treatment. It is suggested that the litchi homologs 18S ribosomal RNA gene, cytochrome P450 like TBP, and the senescence-associated protein, chaperone protein, and a hypothetical protein encoding genes may be involved in the NO-induced senescence in litchi rudimentary leaves. LcERD15-like may be a key gene involved in this process.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

scholarly journals N-Hydroxyarylamine O-Acetyltransferases Catalyze Acetylation of 3-Amino-4-Hydroxyphenylarsonic Acid in the 4-Hydroxy-3-Nitrobenzenearsonic Acid Transformation Pathway of Enterobacter sp. Strain CZ-1

2019 ◽  
Vol 86 (2) ◽  
Author(s):  
Ke Huang ◽  
Fan Gao ◽  
X. Chris Le ◽  
Fang-Jie Zhao
Keyword(s):  
Growth Promotion ◽  
Functional Characterization ◽  
Major Product ◽  
Site Directed Mutagenesis ◽  
Feed Additive ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Arsanilic Acid ◽  
Relative Expression Level

ABSTRACT The organoarsenical feed additive 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone [ROX]) is widely used and released into the environment. We previously showed a two-step pathway of ROX transformation by Enterobacter sp. strain CZ-1 involving the reduction of ROX to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) and the acetylation of 3-AHPAA to N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA) (K. Huang, H. Peng, F. Gao, Q. Liu, et al., Environ Pollut 247:482–487, 2019, https://doi.org/10.1016/j.envpol.2019.01.076). In this study, we identified two nhoA genes (nhoA1 and nhoA2), encoding N-hydroxyarylamine O-acetyltransferases, as responsible for 3-AHPAA acetylation in Enterobacter sp. strain CZ-1. The results of genetic disruption and complementation showed that both nhoA genes are involved in ROX biotransformation and that nhoA1 is the major 3-AHPAA acetyltransferase gene. Quantitative reverse transcription-PCR analysis showed that the relative expression level of nhoA1 was 3-fold higher than that of nhoA2. Each of the recombinant NhoAs was overexpressed in Escherichia coli BL21 and homogenously purified as a dimer by affinity chromatography. Both purified NhoAs catalyzed acetyl coenzyme A-dependent 3-AHPAA acetylation. The Km values of 3-AHPAA for NhoA1 and NhoA2 were 151.5 and 428.3 μM, respectively. Site-directed mutagenesis experiments indicated that two conserved arginine and cysteine residues of each NhoA were necessary for their enzyme activities. IMPORTANCE Roxarsone (ROX) is an organoarsenic feed additive that has been widely used in poultry industries for growth promotion, coccidiosis control, and meat pigmentation improvement for more than 70 years. Most ROX is excreted in the litter and dispersed into the environment, where it is transformed by microbes into different arsenic-containing compounds. A major product of ROX transformation is N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), which is also used as a clinical drug for treating refractory bacterial vaginosis. Here, we report the cloning and functional characterization of two genes encoding N-hydroxyarylamine O-acetyltransferases, NhoA1 and NhoA2, in Enterobacter sp. strain CZ-1, which catalyze the acetylation of 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) formed by the reduction of ROX to N-AHPAA. This study provides new insights into the function of N-hydroxyarylamine O-acetyltransferase in the transformation of an important organoarsenic compound.

scholarly journals CsrA Modulates Levels of Lipoproteins and Key Regulators of Gene Expression Critical for Pathogenic Mechanisms ofBorrelia burgdorferi

2010 ◽  
Vol 79 (2) ◽  
pp. 732-744 ◽  
Author(s):  
S. L. Rajasekhar Karna ◽  
Eva Sanjuan ◽  
Maria D. Esteve-Gassent ◽  
Christine L. Miller ◽  
Mahulena Maruskova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Deletion Mutant ◽  
Rna Binding ◽  
Bacterial Species ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Immunoblot Analysis ◽  
Pcr Analysis ◽  
Mammalian Host ◽  
Reverse Transcription Pcr

ABSTRACTCarbon storage regulator A (CsrA) is an RNA binding protein that has been characterized in many bacterial species to play a central regulatory role by modulating several metabolic processes. We recently showed that a homolog of CsrA inBorrelia burgdorferi(CsrABb, BB0184) was upregulated in response to propagation ofB. burgdorferiunder mammalian host-specific conditions. In order to further delineate the role of CsrABb, we generated a deletion mutant designated ES10 in a linear plasmid 25-negative isolate ofB. burgdorferistrain B31 (ML23). The deletion mutant was screened by PCR and Southern blot hybridization, and a lack of synthesis of CsrABbin ES10 was confirmed by immunoblot analysis. Analysis of ES10 propagated at pH 6.8/37°C revealed a significant reduction in the levels of OspC, DbpA, BBK32, and BBA64 compared to those for the parental wild-type strain propagated under these conditions, while there were no significant changes in the levels of either OspA or P66. Moreover, the levels of two regulatory proteins, RpoS and BosR, were also found to be lower in ES10 than in the control strain. Quantitative real-time reverse transcription-PCR analysis of total RNA extracted from the parental strain andcsrABbmutant revealed significant differences in gene expression consistent with the changes at the protein level. Neither thecsrABbmutant nor thetrans-complemented strain was capable of infection following intradermal needle inoculation in C3H/HeN mice at either 103or 105spirochetes per mouse. The further characterization of molecular basis of regulation mediated by CsrABbwill provide significant insights into the pathophysiology ofB. burgdorferi.

scholarly journals Arr-cb Is a Rifampin Resistance Determinant Found Active or Cryptic in Clostridiumbolteae Strains

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Jean-Christophe Marvaud ◽  
Thierry Lambert
Keyword(s):  
Escherichia Coli ◽  
Gut Microbiota ◽  
Reverse Transcription ◽  
Recent Analysis ◽  
Pcr Analysis ◽  
Directed Mutagenesis ◽  
Human Gut ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Rifampin Resistance

ABSTRACT Clostridium bolteae, which belongs to the Clostridium clostridioforme complex, is a member of the human gut microbiota. Recent analysis of seven genomes of C. bolteae revealed the presence of an arr-like gene. Among these strains, only 90A7 was found to be resistant to rifampin in the absence of alteration of RpoB. Cloning of arr-cb from 90A7 in Escherichia coli combined with directed mutagenesis demonstrated that Arr-cb was functional but that a Q127→R variant present in 90A9 and 90B3 was inactive. Quantitative reverse transcription-PCR analysis indicated that arr-cb was silent in the four remaining strains because of defective transcription. Thus, two independent mechanisms can make the probably intrinsic arr-cb gene of C. bolteae cryptic.

scholarly journals γ-Resorcylate Catabolic-Pathway Genes in the Soil Actinomycete Rhodococcus jostii RHA1

2015 ◽  
Vol 81 (21) ◽  
pp. 7656-7665 ◽  
Author(s):  
Daisuke Kasai ◽  
Naoto Araki ◽  
Kota Motoi ◽  
Shota Yoshikawa ◽  
Toju Iino ◽  
...  
Keyword(s):  
Major Facilitator Superfamily ◽  
Pcr Analysis ◽  
Decarboxylase Activity ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Intergenic Regions ◽  
Major Facilitator Superfamily Transporter ◽  
Maleylacetate Reductase ◽  
Major Facilitator ◽  
Rhodococcus Jostii

ABSTRACTTheRhodococcus jostiiRHA1 gene cluster required for γ-resorcylate (GRA) catabolism was characterized. The cluster includestsdA,tsdB,tsdC,tsdD,tsdR,tsdT, andtsdX, which encode GRA decarboxylase, resorcinol 4-hydroxylase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, an IclR-type regulator, a major facilitator superfamily transporter, and a putative hydrolase, respectively. ThetsdAgene conferred GRA decarboxylase activity onEscherichia coli. Purified TsdB oxidized NADH in the presence of resorcinol, suggesting thattsdBencodes a unique NADH-specific single-component resorcinol 4-hydroxylase. Mutations in eithertsdAortsdBresulted in growth deficiency on GRA. ThetsdCandtsdDgenes conferred hydroxyquinol 1,2-dioxygenase and maleylacetate reductase activities, respectively, onE. coli. Inactivation oftsdTsignificantly retarded the growth of RHA1 on GRA. The growth retardation was partially suppressed under acidic conditions, suggesting the involvement oftsdTin GRA uptake. Reverse transcription-PCR analysis revealed that thetsdgenes constitute three transcriptional units, thetsdBADCandtsdTXoperons andtsdR. Transcription of thetsdBADCandtsdTXoperons was induced during growth on GRA. Inactivation oftsdRderepressed transcription of thetsdBADCandtsdTXoperons in the absence of GRA, suggesting thattsdgene transcription is negatively regulated by thetsdR-encoded regulator. Binding of TsdR to thetsdR-tsdBandtsdT-tsdRintergenic regions was inhibited by the addition of GRA, indicating that GRA interacts with TsdR as an effector molecule.

Characterizing biofilms for biofouling and microbial corrosion control in cooling water systems

2016 ◽  
Vol 63 (6) ◽  
pp. 477-489 ◽  
Author(s):  
R.P. George ◽  
U. Kamachi Mudali ◽  
Baldev Raj
Keyword(s):  
Biofilm Formation ◽  
Corrosion Behaviour ◽  
Control Strategies ◽  
Bacterial Species ◽  
Cooling Water ◽  
Water Systems ◽  
Microbiologically Influenced Corrosion ◽  
Mitigation Measures ◽  
Content Type ◽  
Physiological Diversity

Purpose The purpose of this paper is to study the metal-Microbe interaction playing a crucial role in microbiologically influenced corrosion (MIC) and biofouling of materials in cooling water systems. Treatment regimens should be planned based on this understanding. Design/methodology/approach Attempts were made in the past decades to characterize and understand biofilm formation on important power plant structural materials such as carbon steel (CS), stainless steel (SS) and titanium in fresh water and in seawater to achieve better control of biofouling and minimize MIC problems. Findings This report presents the results of detailed studies on tuberculation-formed CS because of the action of iron-oxidizing bacteria and the effects of algae- and bacteria-dominated biofilms on the passivity of SS. The preferential adhesion of different bacterial species on SS under the influence of inclusions and sensitization was studied in the context of preferential corrosion of SS weldments due to microbial action. Detailed characterization of biofilms formed on titanium (the likely condenser material for fast breeder reactors) after exposure for two years in Kalpakkam coastal waters revealed intense biofouling and biomineralization of manganese even in chlorinated seawater. Studies on the effectiveness of conventional fouling control strategies were also evaluated. Originality/value The detailed studies of different metal/biofilm/microbe interactions demonstrated the physiological diversity of microbes in the biofilms that were formed on different materials, coupling their cooperative metabolic activities with consequent corrosion behaviour. These interactions could enhance either anodic or cathodic reactions and exploit metallurgical features that enhance biofilm formation and/or the capacity of microbes to mutate and overcome mitigation measures.

scholarly journals Bovine Intestinal Bacteria Inactivate and Degrade Ceftiofur and Ceftriaxone with Multiple β-Lactamases

2011 ◽  
Vol 55 (11) ◽  
pp. 4990-4998 ◽  
Author(s):  
R. Doug Wagner ◽  
Shemedia J. Johnson ◽  
Carl E. Cerniglia ◽  
Bruce D. Erickson
Keyword(s):  
High Performance ◽  
Bacterial Species ◽  
Anaerobic Bacteria ◽  
Intestinal Bacteria ◽  
Mass Spectrometry Analysis ◽  
Pcr Analysis ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Similar Drug ◽  
Bacterial Lysates

ABSTRACTThe veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinder-plate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatography-mass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the generaBacillusandBacteroides. PCR analysis of bacterial DNA detected specific β-lactamase genes.Bacillus cereusandB. mycoidesisolates produced extended-spectrum β-lactamases and metallo-β-lactamases. Seven isolates ofBacteroidesspp. produced multiple β-lactamases, including possibly CepA, and metallo-β-lactamases. Isolates ofEubacterium biforme,Bifidobacterium breve, and severalClostridiumspp. also produced ceftiofur-degrading β-lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that β-lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple β-lactamases from bacteria in the large intestines of cattle.

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