scholarly journals Disruption of c-di-GMP signaling networks unlocks cryptic expression of secondary metabolites during biofilm growth in Burkholderia pseudomallei

2021 ◽  
Author(s):  
Grace I Borlee ◽  
Mihnea R. Mangalea ◽  
Kevin H. Martin ◽  
Brooke A. Plumley ◽  
Samuel J. Golon ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Gene Clusters ◽  
Etiologic Agent ◽  
Regulatory Control ◽  
Colony Morphology ◽  
Secretion Systems ◽  
Biofilm Growth ◽  
Conditional Expression

The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei, a soil-borne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at varying temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypo- and hyper- biofilm forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) clusters 2, 11, 14 (syrbactin), and 15 (malleipeptin) in wild-type and ΔII2523 backgrounds also reveals the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani. Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under differing environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions.

scholarly journals Biofilm Formation in Pseudomonas aeruginosa: Fimbrial cup Gene Clusters Are Controlled by the Transcriptional Regulator MvaT

2004 ◽  
Vol 186 (9) ◽  
pp. 2880-2890 ◽  
Author(s):  
Isabelle Vallet ◽  
Stephen P. Diggle ◽  
Rachael E. Stacey ◽  
Miguel Cámara ◽  
Isabelle Ventre ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Dna Microarrays ◽  
Transcriptional Profiling ◽  
Bacterial Pathogen ◽  
Gene Clusters ◽  
Northern Blotting ◽  
Negative Regulator ◽  
Regulatory Control ◽  
Homologous Gene

ABSTRACT Pseudomonas aeruginosa is an opportunistic bacterial pathogen which poses a major threat to long-term-hospitalized patients and individuals with cystic fibrosis. The capacity of P. aeruginosa to form biofilms is an important requirement for chronic colonization of human tissues and for persistence in implanted medical devices. Various stages of biofilm formation by this organism are mediated by extracellular appendages, such as type IV pili and flagella. Recently, we identified three P. aeruginosa gene clusters that were termed cup (chaperone-usher pathway) based on their sequence relatedness to the chaperone-usher fimbrial assembly pathway in other bacteria. The cupA gene cluster, but not the cupB or cupC cluster, is required for biofilm formation on abiotic surfaces. In this study, we identified a gene (mvaT) encoding a negative regulator of cupA expression. Such regulatory control was confirmed by several approaches, including lacZ transcriptional fusions, Northern blotting, and transcriptional profiling using DNA microarrays. MvaT also represses the expression of the cupB and cupC genes, although the extent of the regulatory effect is not as pronounced as with cupA. Consistent with this finding, mvaT mutants exhibit enhanced biofilm formation. Although the P. aeruginosa genome contains a highly homologous gene, mvaU, the repression of cupA genes is MvaT specific. Thus, MvaT appears to be an important regulatory component within a complex network that controls biofilm formation and maturation in P. aeruginosa.

scholarly journals Genetic Characterization of Pseudomonas fluorescens SBW25 rsp Gene Expression in the Phytosphere and In Vitro

2005 ◽  
Vol 187 (24) ◽  
pp. 8477-8488 ◽  
Author(s):  
Robert W. Jackson ◽  
Gail M. Preston ◽  
Paul B. Rainey
Keyword(s):  
Pseudomonas Fluorescens ◽  
Ectopic Expression ◽  
Gene Clusters ◽  
Regulatory Genes ◽  
Regulatory Control ◽  
Lag Phase ◽  
Secretion Systems ◽  
General Role ◽  
Animal Pathogens

ABSTRACT The plant-colonizing Pseudomonas fluorescens strain SBW25 harbors a gene cluster (rsp) whose products show similarity to type III protein secretion systems found in plant and animal pathogens. Here we report a detailed analysis of the expression and regulation of the P. fluorescens rsp pathway, both in the phytosphere and in vitro. A combination of chromosomally integrated transcriptional reporter fusions, overexpressed regulatory genes, and specific mutants reveal that promoters controlling expression of rsp are actively transcribed in the plant rhizosphere but not (with the exception of the rspC promoter) in the phyllosphere. In synthetic medium, regulatory (rspL and rspR) and structural (rspU, plus the putative effector ropE) genes are poorly expressed; the rspC promoter is subject to an additional level of regulatory control. Ectopic expression of regulatory genes in wild-type and mutant backgrounds showed that RspR controls transcription of the alternate sigma factor, rspL, and that RspL controls expression of gene clusters encoding structural genes. Mutation of rspV did not affect RspR-mediated expression of rspU. A search for additional regulators revealed two candidates—one with a role in the conversion of alanine to pyruvate—suggesting that expression of rsp is partly dependent upon the metabolic status of the cell. Mutations in rsp regulators resulted in a significant reduction in competitive colonization of the root tips of sugar beet seedlings but also caused a marked increase in the lag phase of laboratory-grown cultures, indicating that rsp regulatory genes play a more significant general role in the function of P. fluorescens SBW25 than previously appreciated.

scholarly journals An integrated model system to gain mechanistic insights into biofilm formation and antimicrobial resistance development in Pseudomonas aeruginosa MPAO1

2020 ◽  
Author(s):  
Adithi R. Varadarajan ◽  
Raymond N. Allan ◽  
Jules D. P. Valentin ◽  
Olga E. Castañeda Ocampo ◽  
Vincent Somerville ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Soft Lithography ◽  
Flow Chamber ◽  
Model System ◽  
Secretion Systems ◽  
Phenotypic Data ◽  
Biofilm Growth ◽  
Mutant Collection

AbstractPseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and antibiotic resistance.Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and missed genes by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq datasets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth. Experiments conducted across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated both known and novel genes involved in biofilm growth and antibiotic resistance identified in screens of the mutant collection. Differential protein expression data from planktonic cells versus biofilm confirmed upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type six secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance and resistance evolution.

scholarly journals Genetic requirements and transcriptomics of Helicobacter pylori biofilm formation on abiotic and biotic surfaces

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Skander Hathroubi ◽  
Shuai Hu ◽  
Karen M. Ottemann
Keyword(s):  
Helicobacter Pylori ◽  
Biofilm Formation ◽  
Tca Cycle ◽  
Harsh Environments ◽  
Laboratory Model ◽  
Secretion Systems ◽  
Biofilm Growth ◽  
Tca Cycle Enzymes ◽  
Growth Properties ◽  
H Pylori

AbstractBiofilm growth is a widespread mechanism that protects bacteria against harsh environments, antimicrobials, and immune responses. These types of conditions challenge chronic colonizers such as Helicobacter pylori but it is not fully understood how H. pylori biofilm growth is defined and its impact on H. pylori survival. To provide insights into H. pylori biofilm growth properties, we characterized biofilm formation on abiotic and biotic surfaces, identified genes required for biofilm formation, and defined the biofilm-associated gene expression of the laboratory model H. pylori strain G27. We report that H. pylori G27 forms biofilms with a high biomass and complex flagella-filled 3D structures on both plastic and gastric epithelial cells. Using a screen for biofilm-defective mutants and transcriptomics, we discovered that biofilm cells demonstrated lower transcripts for TCA cycle enzymes but higher ones for flagellar formation, two type four secretion systems, hydrogenase, and acetone metabolism. We confirmed that biofilm formation requires flagella, hydrogenase, and acetone metabolism on both abiotic and biotic surfaces. Altogether, these data suggest that H. pylori is capable of adjusting its phenotype when grown as biofilm, changing its metabolism, and re-shaping flagella, typically locomotion organelles, into adhesive structures.

scholarly journals Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

2014 ◽  
Vol 81 (1) ◽  
pp. 422-431 ◽  
Author(s):  
Chuping Luo ◽  
Xuehui Liu ◽  
Huafei Zhou ◽  
Xiaoyu Wang ◽  
Zhiyi Chen
Keyword(s):  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Hemolytic Activity ◽  
Biofilm Formation ◽  
Gene Clusters ◽  
Colony Morphology ◽  
Swarming Motility ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Phenotypic Features

ABSTRACTBacilluscyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features ofBacillusstrains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology.Bacillus subtilis916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome ofB. subtilis916 contains four nonribosomal peptide synthase (NRPS) gene clusters,srf,bmy,fen, andloc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studyingB. subtilis916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activityin vitro, the strain mutated insrfAAhad significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other thanfenresulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion,B. subtilis916 coproduces four families of LPs which contribute to the phenotypic features ofB. subtilis916 in an intricate way.

scholarly journals An integrated model system to gain mechanistic insights into biofilm-associated antimicrobial resistance in Pseudomonas aeruginosa MPAO1

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Adithi R. Varadarajan ◽  
Raymond N. Allan ◽  
Jules D. P. Valentin ◽  
Olga E. Castañeda Ocampo ◽  
Vincent Somerville ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Soft Lithography ◽  
Flow Chamber ◽  
Model System ◽  
Secretion Systems ◽  
Phenotypic Data ◽  
Biofilm Growth ◽  
Resistance Evolution

Abstract Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.

scholarly journals Role of HrpA in Biofilm Formation of Neisseria meningitidis and Regulation of the hrpBAS Transcripts

2009 ◽  
Vol 77 (6) ◽  
pp. 2285-2293 ◽  
Author(s):  
R. Brock Neil ◽  
Michael A. Apicella
Keyword(s):  
Neisseria Meningitidis ◽  
Biofilm Formation ◽  
Cell Contact ◽  
Wild Type ◽  
Secretion Systems ◽  
Biofilm Growth ◽  
Bronchial Epithelial ◽  
Gram Negative Organisms ◽  
Biofilm Structure

ABSTRACT Two-partner secretion systems of gram-negative organisms are utilized in adherence, invasion, and biofilm formation. The HrpAB proteins of Neisseria meningitidis are members of a two-partner secretion system, and HrpA is established as being important to adherence and intracellular escape. This study set out to determine the expression pattern of members of the hrpBAS putative operon and to find a functional role for the HrpA protein. The upregulation of these genes was found in situations of anaerobiosis and cell contact. These observations prompted the study of the function of HrpA in biofilms on human bronchial epithelial cells. HrpA mutants in encapsulated and unencapsulated NMB strains demonstrated biofilm growth equivalent to that of the wild-type strain at 6 h but a decreased ability to form biofilms at 48 h. Biofilms formed by hrpA mutants for 48 h on collagen-coated coverslips demonstrated significant reductions compared to those of wild-type strains. Taken together, these observations imply a role for HrpA in the biofilm structure. Further analysis demonstrated the presence of HrpA on the surface of the bacterium.

scholarly journals Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

2014 ◽  
Vol 82 (8) ◽  
pp. 3214-3226 ◽  
Author(s):  
Mary N. Burtnick ◽  
Paul J. Brett ◽  
David DeShazer
Keyword(s):  
Burkholderia Pseudomallei ◽  
Signal Sequence ◽  
Hydrolytic Enzymes ◽  
Opportunistic Pathogen ◽  
Etiologic Agent ◽  
Type Ii ◽  
Secretion Systems ◽  
Content Type

ABSTRACTBurkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provideB. pseudomalleiwith a growth advantagein vitroandin vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations ingspDandgspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles ofB. pseudomalleiMSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants ofB. pseudomalleiMSHR668 andB. pseudomalleiΔgspDgrown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ΔgspDstrains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of theB. pseudomalleiT2SS secretome.

scholarly journals Thermoregulation of Biofilm Formation in Burkholderia pseudomallei Is Disrupted by Mutation of a Putative Diguanylate Cyclase

2016 ◽  
Vol 199 (5) ◽  
Author(s):  
Brooke A. Plumley ◽  
Kevin H. Martin ◽  
Grace I. Borlee ◽  
Nicole L. Marlenee ◽  
Mary N. Burtnick ◽  
...  
Keyword(s):  
Environmental Conditions ◽  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Etiologic Agent ◽  
Mammalian Host ◽  
Insertion Mutant ◽  
Diguanylate Cyclase ◽  
Content Type ◽  
Transposon Insertion ◽  
Tier 1

ABSTRACT Burkholderia pseudomallei, a tier 1 select agent and the etiological agent of melioidosis, transitions from soil and aquatic environments to infect a variety of vertebrate and invertebrate hosts. During the transition from an environmental saprophyte to a mammalian pathogen, B. pseudomallei encounters and responds to rapidly changing environmental conditions. Environmental sensing systems that control cellular levels of cyclic di-GMP promote pathogen survival in diverse environments. Cyclic di-GMP controls biofilm production, virulence factors, and motility in many bacteria. This study is an evaluation of cyclic di-GMP-associated genes that are predicted to metabolize and interact with cyclic di-GMP as identified from the annotated genome of B. pseudomallei 1026b. Mutants containing transposon disruptions in each of these genes were characterized for biofilm formation and motility at two temperatures that reflect conditions that the bacteria encounter in the environment and during the infection of a mammalian host. Mutants with transposon insertions in a known phosphodiesterase (cdpA) and a predicted hydrolase (Bp1026b_I2285) gene exhibited decreased motility regardless of temperature. In contrast, the phenotypes exhibited by mutants with transposon insertion mutations in a predicted diguanylate cyclase gene (Bp1026b_II2523) were strikingly influenced by temperature and were dependent on a conserved GG(D/E)EF motif. The transposon insertion mutant exhibited enhanced biofilm formation at 37°C but impaired biofilm formation at 30°C. These studies illustrate the importance of studying behaviors regulated by cyclic di-GMP under varied environmental conditions in order to better understand cyclic di-GMP signaling in bacterial pathogens. IMPORTANCE This report evaluates predicted cyclic di-GMP binding and metabolic proteins from Burkholderia pseudomallei 1026b, a tier 1 select agent and the etiologic agent of melioidosis. Transposon insertion mutants with disruptions in each of the genes encoding these predicted proteins were characterized in order to identify key components of the B. pseudomallei cyclic di-GMP-signaling network. A predicted hydrolase and a phosphodiesterase that modulate swimming motility were identified, in addition to a diguanylate cyclase that modulates biofilm formation and motility in response to temperature. These studies warrant further evaluation of the contribution of cyclic di-GMP to melioidosis in the context of pathogen acquisition from environmental reservoirs and subsequent colonization, dissemination, and persistence within the host.

Genome sequence of Novosphingobium malaysiense strain MUSC 273T, novel alpha-proteobacterium isolated from intertidal soil

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Hooi-Leng Ser ◽  
Wai-Fong Yin ◽  
Kok-Gan Chan ◽  
Nurul-Syakima Ab Mutalib ◽  
Learn-Han Lee
Keyword(s):  
Secondary Metabolites ◽  
Secondary Metabolite ◽  
Genome Sequence ◽  
Catalase Activity ◽  
Gene Clusters ◽  
Rrna Genes ◽  
Gram Negative ◽  
Genomic Features

Novosphingobium malaysiense strain MUSC 273T is a recently identified Gram-negative, aerobic alpha-proteobacterium. The strain was isolated from intertidal soil with strong catalase activity. The genome sequence comprises 5,027,021 bp, with 50 tRNA and 3 rRNA genes. Further analysis identified presence of secondary metabolite gene clusters within genome of MUSC 273T. Knowledge of the genomic features of the strain may allow further biotechnological exploitation, particularly for production of secondary metabolites as well as production of industrially important enzymes

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