scholarly journals Species-Specific Interactions of Arr with RplK Mediate Stringent Response in Bacteria

2018 ◽  
Vol 200 (6) ◽  
Author(s):  
Priyanka Agrawal ◽  
Rajagopal Varada ◽  
Shivjee Sah ◽  
Souvik Bhattacharyya ◽  
Umesh Varshney
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Stringent Response ◽  
Content Type ◽  
E Coli ◽  
Fitness Advantage ◽  
Species Specific ◽  
Ribosomal Protein L11

ABSTRACTBacteria respond to stressful growth conditions through a conserved phenomenon of stringent response mediated by synthesis of stress alarmones ppGpp and pppGpp [referred to as (p)ppGpp]. (p)ppGpp synthesis is known to occur by ribosome-associated RelA. In addition, a dual-function protein, SpoT (with both synthetase and hydrolase activities), maintains (p)ppGpp homeostasis. The presence of (p)ppGpp is also known to contribute to antibiotic resistance in bacteria.Mycobacterium smegmatispossesses Arr, which inactivates rifampin by its ADP ribosylation. Arr has been shown to be upregulated in response to stress. However, the roles Arr might play during growth have remained unclear. We show that Arr confers growth fitness advantage toM. smegmatiseven in the absence of rifampin. Arr deficiency inM. smegmatisresulted in deficiency of biofilm formation. Further, we show that while Arr does not interact with the wild-typeEscherichia coliribosomes, it interacts with them when theE. coliribosomal protein L11 (a stringent response regulator) is replaced with its homolog fromM. smegmatis. The Arr interaction withE. coliribosomes occurs even when the N-terminal 33 amino acids of its L11 protein were replaced with the corresponding sequence ofM. smegmatisL11 (Msm-EcoL11 chimeric protein). Interestingly, Arr interaction with theE. coliribosomes harboringM. smegmatisL11 orMsm-EcoL11 results in the synthesis of ppGppin vivo. Our study shows a novel role of antibiotic resistance genearrin stress response.IMPORTANCEMycobacterium smegmatis, like many other bacteria, possesses an ADP-ribosyltransferase, Arr, which confers resistance to the first-line antituberculosis drug, rifampin, by its ADP ribosylation. In this report, we show that in addition to its known property of conferring resistance to rifampin, Arr confers growth fitness advantage toM. smegmatiseven when there is no rifampin in the growth medium. We then show that Arr establishes species-specific interactions with ribosomes through the N-terminal sequence of ribosomal protein L11 (a stringent response regulator) and results in ppGpp (stress alarmone) synthesis. Deficiency of Arr inM. smegmatisresults in deficiency of biofilm formation. Arr protein is physiologically important both in conferring antibiotic resistance as well as in mediating stringent response.

scholarly journals The OmpR Regulator ofBurkholderia multivoransControls Mucoid-to-Nonmucoid Transition and Other Cell Envelope Properties Associated with Persistence in the Cystic Fibrosis Lung

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Inês N. Silva ◽  
Filipa D. Pessoa ◽  
Marcelo J. Ramires ◽  
Mário R. Santos ◽  
Jörg D. Becker ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Respiratory Infections ◽  
Response Regulator ◽  
Cell Envelope ◽  
Content Type ◽  
Burkholderia Multivorans ◽  
Exopolysaccharide Production ◽  
High Osmolarity

ABSTRACTBacteria from theBurkholderia cepaciacomplex grow in different natural and man-made environments and are feared opportunistic pathogens that cause chronic respiratory infections in cystic fibrosis patients. Previous studies showed thatBurkholderiamucoid clinical isolates grown under stress conditions give rise to nonmucoid variants devoid of the exopolysaccharide cepacian. Here, we determined that a major cause of the nonmucoid morphotype involves nonsynonymous mutations and small indels in theompRgene encoding a response regulator of a two-component regulatory system. Intranscomplementation of nonmucoid variants (NMVs) with the native gene restored exopolysaccharide production. The loss of functionalBurkholderia multivoransOmpR had positive effects on growth, adhesion to lung epithelial cells, and biofilm formation in high-osmolarity medium, as well as an increase in swimming and swarming motilities. In contrast, phenotypes such as antibiotic resistance, biofilm formation at low osmolarity, and virulence inGalleria mellonellawere compromised by the absence of functional OmpR. Transcriptomic studies indicated that loss of theompRgene affects the expression of 701 genes, many associated with outer membrane composition, motility, stress response, iron acquisition, and the uptake of nutrients, consistent with starvation tolerance. Since the stresses here imposed onB. multivoransmay strongly resemble the ones found in the cystic fibrosis (CF) airways and mutations in theompRgene from longitudinally collected CF isolates have been found, this regulator might be important for the production of NMVs in the CF environment.IMPORTANCEWithin the cystic fibrosis (CF) lung, bacteria experience high-osmolarity conditions due to an ion unbalance resulting from defects in CF transmembrane conductance regulator (CFTR) protein activity in epithelial cells. Understanding how bacterial CF pathogens thrive in this environment might help the development of new therapeutic interventions to prevent chronic respiratory infections. Here, we show that the OmpR response regulator of one of the species found in CF respiratory infections,Burkholderia multivorans, is involved in the emergence of nonmucoid colony variants and is important for osmoadaptation by regulating several cell envelope components. Specifically, genetic, phenotypic, genomic, and transcriptomic approaches uncover OmpR as a regulator of cell wall remodeling under stress conditions, with implications in several phenotypes such as exopolysaccharide production, motility, antibiotic resistance, adhesion, and virulence.

scholarly journals The Stringent Response Regulator DksA Is Required for Salmonella enterica Serovar Typhimurium Growth in Minimal Medium, Motility, Biofilm Formation, and Intestinal Colonization

2015 ◽  
Vol 84 (1) ◽  
pp. 375-384 ◽  
Author(s):  
Shalhevet Azriel ◽  
Alina Goren ◽  
Galia Rahav ◽  
Ohad Gal-Mor
Keyword(s):  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Early Stage ◽  
Response Regulator ◽  
Stringent Response ◽  
Negative Regulator ◽  
Logarithmic Phase ◽  
Content Type ◽  
Serovar Typhimurium

Salmonella entericaserovar Typhimurium is a facultative intracellular human and animal bacterial pathogen posing a major threat to public health worldwide.Salmonellapathogenicity requires complex coordination of multiple physiological and virulence pathways. DksA is a conserved Gram-negative regulator that belongs to a distinct group of transcription factors that bind directly to the RNA polymerase secondary channel, potentiating the effect of the signaling molecule ppGpp during a stringent response. Here, we established that inS.Typhimurium,dksAis induced during the logarithmic phase and DksA is essential for growth in minimal defined medium and plays an important role in motility and biofilm formation. Furthermore, we determined that DksA positively regulates theSalmonellapathogenicity island 1 and motility-chemotaxis genes and is necessary forS.Typhimurium invasion of human epithelial cells and uptake by macrophages. In contrast, DksA was found to be dispensable forS.Typhimurium host cell adhesion. Finally, using the colitis mouse model, we found thatdksAis spatially induced at the midcecum during the early stage of the infection and required for gastrointestinal colonization and systemic infectionin vivo. Taken together, these data indicate that the ancestral stringent response regulator DksA coordinates various physiological and virulenceS.Typhimurium programs and therefore is a key virulence regulator ofSalmonella.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Reed M. Stubbendieck ◽  
Paul D. Straight
Keyword(s):  
Antibiotic Resistance ◽  
Abc Transporter ◽  
Bacterial Communities ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Bioactive Metabolites ◽  
Signaling System ◽  
Content Type ◽  
Two Component ◽  
Analytical Approaches

ABSTRACT Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilis. IMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that includes the gastrointestinal tract of animals and the rhizosphere of plants. Interactions between different species in bacterial communities have impacts on our health and industry. However, many approaches currently used to study whole bacterial communities do not resolve mechanistic details of interspecies interactions, including how bacteria sense and respond to their competitors. Using a competition model, we have uncovered dual functions for a previously uncharacterized two-component signaling system involved in specific antibiotic resistance and biofilm morphology. Insights gleaned from signaling within interspecies interaction models build a more complete understanding of gene functions important for bacterial communities and will enhance community-level analytical approaches.

scholarly journals Increased Survival of Antibiotic-Resistant Escherichia coli inside Macrophages

2012 ◽  
Vol 57 (1) ◽  
pp. 189-195 ◽  
Author(s):  
Migla Miskinyte ◽  
Isabel Gordo
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Bacterial Infections ◽  
Resistance Mutation ◽  
Fitness Cost ◽  
Resistance Mutations ◽  
Content Type ◽  
E Coli ◽  
Fitness Effects ◽  
K 12

ABSTRACTMutations causing antibiotic resistance usually incur a fitness cost in the absence of antibiotics. The magnitude of such costs is known to vary with the environment. Little is known about the fitness effects of antibiotic resistance mutations when bacteria confront the host's immune system. Here, we study the fitness effects of mutations in therpoB,rpsL, andgyrAgenes, which confer resistance to rifampin, streptomycin, and nalidixic acid, respectively. These antibiotics are frequently used in the treatment of bacterial infections. We measured two important fitness traits—growth rate and survival ability—of 12Escherichia coliK-12 strains, each carrying a single resistance mutation, in the presence of macrophages. Strikingly, we found that 67% of the mutants survived better than the susceptible bacteria in the intracellular niche of the phagocytic cells. In particular, allE. colistreptomycin-resistant mutants exhibited an intracellular advantage. On the other hand, 42% of the mutants incurred a high fitness cost when the bacteria were allowed to divide outside of macrophages. This study shows that single nonsynonymous changes affecting fundamental processes in the cell can contribute to prolonged survival ofE. coliin the context of an infection.

scholarly journals Pumilacidin-Like Lipopeptides Derived from Marine Bacterium Bacillus sp. Strain 176 Suppress the Motility of Vibrio alginolyticus

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Pengyuan Xiu ◽  
Rui Liu ◽  
Dechao Zhang ◽  
Chaomin Sun
Keyword(s):  
Cell Death ◽  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Marine Bacterium ◽  
Vibrio Alginolyticus ◽  
Bacterial Motility ◽  
Great Promise ◽  
Content Type ◽  
Cyclic Lipopeptides

ABSTRACT Bacterial motility is a crucial factor during the invasion and colonization processes of pathogens, which makes it an attractive therapeutic drug target. Here, we isolated a marine bacterium (Vibrio alginolyticus strain 178) from a seamount in the tropical West Pacific that exhibits vigorous motility on agar plates and severe pathogenicity to zebrafish. We found that V. alginolyticus 178 motility was significantly suppressed by another marine bacterium, Bacillus sp. strain 176, isolated from the same niche. We isolated, purified, and characterized two different cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The two related CLPs have a pumilacidin-like structure and were both effective inhibitors of V. alginolyticus 178 motility. The CLPs differ by only one methylene group in their fatty acid chains. In addition to motility suppression, the CLPs also induced cell aggregation in the medium and reduced adherence of V. alginolyticus 178 to glass substrates. Notably, upon CLP treatment, the expression levels of two V. alginolyticus flagellar assembly genes (flgA and flgP) dropped dramatically. Moreover, the CLPs inhibited biofilm formation in several other strains of pathogenic bacteria without inducing cell death. This study indicates that CLPs from Bacillus sp. 176 show promise as antimicrobial lead compounds targeting bacterial motility and biofilm formation with a low potential for eliciting antibiotic resistance. IMPORTANCE Pathogenic bacteria often require motility to establish infections and subsequently spread within host organisms. Thus, motility is an attractive therapeutic target for the development of novel antibiotics. We found that cyclic lipopeptides (CLPs) produced by marine bacterium Bacillus sp. strain 176 dramatically suppress the motility of the pathogenic bacterium Vibrio alginolyticus strain 178, reduce biofilm formation, and promote cellular aggregation without inducing cell death. These findings suggest that CLPs hold great promise as potential drug candidates targeting bacterial motility and biofilm formation with a low overall potential for triggering antibiotic resistance.

scholarly journals Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

scholarly journals Bioinformatic and Molecular Analysis of Inverse Autotransporters from Escherichia coli

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Kelvin G. K. Goh ◽  
Danilo G. Moriel ◽  
Steven J. Hancock ◽  
Minh-Duy Phan ◽  
Mark A. Schembri
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Secretion System ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Type V Secretion ◽  
K 12 ◽  
Type V

ABSTRACT Proteins secreted by the type V secretion system possess multiple functions, including the capacity to mediate adhesion, aggregation, and biolfilm formation. The type V secretion system can be divided into five subclasses, one of which is the type Ve system. Proteins of the type Ve secretion system are also referred to as inverse autotransporters (IATs). In this study, we performed an in silico analysis of 126 completely sequenced Escherichia coli genomes available in the NCBI database and identified several distinct IAT-encoding gene families whose distribution varied throughout the E. coli phylogeny. The genes included three characterized IATs (intimin, fdeC, and yeeJ) and four uncharacterized IATs (here named iatA, iatB, iatC, and iatD). The four iat genes were cloned from the completely sequenced environmental E. coli strain SMS-3-5 and characterized. Three of these IAT proteins (IatB, IatC, and IatD) were expressed at the cell surface and possessed the capacity to mediate biofilm formation in a recombinant E. coli K-12 strain. Further analysis of the iatB gene, which showed a unique association with extraintestinal E. coli strains, suggested that its regulation is controlled by the LeuO global regulator. Overall, this study provides new data describing the prevalence, sequence variation, domain structure, function, and regulation of IATs found in E. coli. IMPORTANCE Escherichia coli is one of the most prevalent facultative anaerobes of the human gut. E. coli normally exists as a harmless commensal but can also cause disease following the acquisition of genes that enhance its pathogenicity. Adhesion is an important first step in colonization of the host and is mediated by an array of cell surface components. In E. coli, these include a family of adhesins secreted by the type V secretion system. Here, we identified and characterized new proteins from an emerging subclass of the type V secretion system known as the inverse autotransporters (IATs). We found that IAT-encoding genes are present in a wide range of strains and showed that three novel IATs were localized on the E. coli cell surface and mediated biofilm formation. Overall, this study provides new insight into the prevalence, function, and regulation of IATs in E. coli.

CRISPR-Cas9 knockout of qseB induced asynchrony between motility and biofilm formation in Escherichia coli

2019 ◽  
Vol 65 (9) ◽  
pp. 691-702 ◽  
Author(s):  
Yi Gou ◽  
Weiqi Liu ◽  
Jing Jing Wang ◽  
Ling Tan ◽  
Bin Hong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Motility ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Signal Transmission ◽  
Bacterial Motility ◽  
Type I ◽  
E Coli ◽  
Real Time Quantitative Pcr ◽  
In The Wild

Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) serves as a bridge for bacterial signal transmission, in which the protein QseB acts as a response regulator bacterial motility, biofilm formation, and virulence. The mechanisms that govern the interaction between QseBC and their functions have been studied in general, but the regulatory role of QseB on bacterial motility and biofilm formation is unknown. In this study, the CRISPR-Cas9 system was used to construct the Escherichia coli MG1655ΔqseB strain (strain ΔqseB), and the effects of the qseB gene on changes in motility and biofilm formation in the wild type (WT) were determined. The motility assay results showed that the ΔqseB strain had higher (p < 0.05) motility than the WT strain. However, there was no difference in the formation of biofilm between the ΔqseB and WT strains. Real-time quantitative PCR illustrated that deletion of qseB in the WT strain downregulated expression of the type I pili gene fimA. Therefore, we might conclude that the ΔqseB induced the downregulation of fimA, which led to asynchrony between motility and biofilm formation in E. coli, providing new insight into the functional importance of QseB in regulating cell motility and biofilm formation.

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