Genetic and Functional Characterization of Cyclic Lipopeptide White-Line-Inducing Principle (WLIP) Production by Rice Rhizosphere Isolate Pseudomonas putida RW10S2

ABSTRACTThe secondary metabolite mediating the GacS-dependent growth-inhibitory effect exerted by the rice rhizosphere isolatePseudomonas putidaRW10S2 on phytopathogenicXanthomonasspecies was identified as white-line-inducing principle (WLIP), a member of the viscosin group of cyclic lipononadepsipeptides. WLIP producers are commonly referred to by the taxonomically invalid name “Pseudomonas reactans,” based on their capacity to reveal the presence of a nearby colony ofPseudomonas tolaasiiby inducing the formation of a visible precipitate (“white line”) in agar medium between both colonies. This phenomenon is attributed to the interaction of WLIP with a cyclic lipopeptide of a distinct structural group, the fungitoxic tolaasin, and has found application as a diagnostic tool to identify tolaasin-producing bacteria pathogenic to mushrooms. The genes encoding the WLIP nonribosomal peptide synthetases WlpA, WlpB, and WlpC were identified in two separate genomic clusters (wlpR-wlpAandwlpBC) with an operon organization similar to that of the viscosin, massetolide, and entolysin biosynthetic systems. Expression ofwlpRis dependent ongacS, and the encoded regulator of the LuxR family (WlpR) activates transcription of the biosynthetic genes and the linked export genes, which is not controlled by the RW10S2 quorum-sensing system PmrR/PmrI. In addition to linking the known phenotypes of white line production and hemolytic activity of a WLIP producer with WLIP biosynthesis, additional properties of ecological relevance conferred by WLIP production were identified, namely, antagonism againstXanthomonasand involvement in swarming and biofilm formation.

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Small-Molecule Inhibition of Choline Catabolism in Pseudomonas aeruginosa and Other Aerobic Choline-Catabolizing Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00504-11 ◽

2011 ◽

Vol 77 (13) ◽

pp. 4383-4389 ◽

Cited By ~ 12

Author(s):

Liam F. Fitzsimmons ◽

Stevenson Flemer ◽

A. Sandy Wurthmann ◽

P. Bruce Deker ◽

Indra Neil Sarkar ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Burkholderia Cepacia ◽

Sinorhizobium Meliloti ◽

Growth Inhibitor ◽

Bioinformatic Analysis ◽

Content Type ◽

Genes Encoding ◽

Dependent Growth

ABSTRACTCholine is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolismin vitroandin situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation inPseudomonas aeruginosa. We used genetic analyses and13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor inP. aeruginosabut did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, includingPseudomonas mendocina,Pseudomonas fluorescens,Pseudomonas putida,Burkholderia cepacia,Burkholderia ambifaria, andSinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolismin situ.

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Effects of Three Different Nucleoid-Associated Proteins Encoded on IncP-7 Plasmid pCAR1 on Host Pseudomonas putida KT2440

Applied and Environmental Microbiology ◽

10.1128/aem.00023-15 ◽

2015 ◽

Vol 81 (8) ◽

pp. 2869-2880 ◽

Cited By ~ 14

Author(s):

Chiho Suzuki-Minakuchi ◽

Ryusuke Hirotani ◽

Masaki Shintani ◽

Toshiharu Takeda ◽

Yurika Takahashi ◽

...

Keyword(s):

Pseudomonas Putida ◽

Structural Instability ◽

Metabolic Phenotype ◽

Pseudomonas Putida Kt2440 ◽

Host Chromosome ◽

Content Type ◽

Inorganic Ion ◽

Segregational Stability ◽

Genes Encoding ◽

Associated Proteins

ABSTRACTNucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption ofpmr,pnd, andphuwere assessed in hostPseudomonas putidaKT2440. Whenpmrandpndorpmrandphuwere simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation ofpmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

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Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida

Applied and Environmental Microbiology ◽

10.1128/aem.03236-16 ◽

2017 ◽

Vol 83 (7) ◽

Cited By ~ 19

Author(s):

Klara Bojanovič ◽

Isotta D'Arrigo ◽

Katherine S. Long

Keyword(s):

Gene Expression ◽

Stress Response ◽

Pseudomonas Putida ◽

Cellular Response ◽

Functional Characterization ◽

Transcriptional Response ◽

Stress Conditions ◽

Differentially Expressed ◽

Transcriptional Responses ◽

Content Type

ABSTRACTBacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response ofPseudomonas putidaKT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through whichP. putidaresponds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings.IMPORTANCEThis study maps the complete transcriptional response ofP. putidaKT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization.

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Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86

Applied and Environmental Microbiology ◽

10.1128/aem.03326-16 ◽

2017 ◽

Vol 83 (8) ◽

Cited By ~ 12

Author(s):

Karishma Mohan ◽

Prashant S. Phale

Keyword(s):

Carbon Source ◽

Ferulic Acid ◽

Pseudomonas Putida ◽

Vanillic Acid ◽

Veratryl Alcohol ◽

Veratric Acid ◽

Suitable Candidate ◽

Content Type ◽

Metabolic Intermediates ◽

Genes Encoding

ABSTRACT Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz., veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz., VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products. IMPORTANCE Pseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the metabolic pathway and gene pool for lignin degradation in bacteria. The biochemical and genetic characterization of phenylpropanoid metabolism makes it a prospective system for its application in producing valuable products, such as vanillin and vanillic acid, from lignocellulose. This study supports the immense potential of P. putida CSV86 as a suitable candidate for bioremediation and biorefinery.

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Draft Genome Sequence of Pseudomonas aeruginosa Strain LMG 1272, an Atypical White Line Reaction Producer

Microbiology Resource Announcements ◽

10.1128/mra.01363-19 ◽

2020 ◽

Vol 9 (7) ◽

Author(s):

Farzaneh Salari ◽

Fatemeh Zare-Mirakabad ◽

Mohammad Hossein Alavi ◽

Léa Girard ◽

Mahya Ghafari ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Genome Sequence ◽

Draft Genome ◽

Draft Genome Sequence ◽

Line Formation ◽

White Line ◽

Pseudomonas Tolaasii ◽

Content Type ◽

Cyclic Lipopeptide ◽

Diffusible Factor

The draft genome sequence of Pseudomonas aeruginosa LMG 1272, isolated from mushroom, is reported here. This strain triggers formation of a precipitate (“white line”) when cocultured with Pseudomonas tolaasii. However, LMG 1272 lacks the capacity to produce a cyclic lipopeptide that is typically associated with white line formation, suggesting the involvement of a different diffusible factor.

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Functional Characterization of the Quorum Sensing Regulator RsaL in the Plant-Beneficial Strain Pseudomonas putida WCS358

Applied and Environmental Microbiology ◽

10.1128/aem.06442-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 726-734 ◽

Cited By ~ 8

Author(s):

Giordano Rampioni ◽

Iris Bertani ◽

Cejoice Ramachandran Pillai ◽

Vittorio Venturi ◽

Elisabetta Zennaro ◽

...

Keyword(s):

Quorum Sensing ◽

Plant Growth ◽

Pseudomonas Putida ◽

Plant Pathogens ◽

Functional Characterization ◽

Signal Molecule ◽

Content Type ◽

Repressive Effect ◽

Plant Growth Promoting ◽

Growth Promoting

ABSTRACTIn many bacteria, quorum sensing (QS) systems rely on a signal receptor and a synthase producingN-acyl-homoserine lactone(s) as the signal molecule(s). In some species, thersaLgene, located between the signal receptor and synthase genes, encodes a repressor limiting signal synthase expression and hence signal molecule production. Here we investigate the molecular mechanism of action of the RsaL protein in the plant growth-promoting rhizobacteriumPseudomonas putidaWCS358 (RsaLWCS). InP. putidaWCS358, RsaLWCSdisplayed a strong repressive effect on the promoter of the QS signal synthase gene,ppuI, while it did not repress the same promoter inPseudomonas aeruginosa. DNase I protection assays showed that purified RsaLWCSspecifically binds toppuIon a DNA region overlapping the predicted σ70-binding site, but such protection was observed only at high protein concentrations. Accordingly, electrophoretic mobility shift assays showed that the RsaLWCSprotein was not able to form stable complexes efficiently with a probe encompassing theppuIpromoter, while it formed stable complexes with the promoter oflasI, the gene orthologous toppuIinP. aeruginosa. This difference seems to be dictated by the lower dyad symmetry of the RsaLWCS-binding sequence on theppuIpromoter relative to that on thelasIpromoter. Comparison of the results obtainedin vivoandin vitrosuggests that RsaLWCSneeds a molecular interactor/cofactor specific forP. putidaWCS358 to repressppuItranscription. We also demonstrate that RsaLWCSregulates siderophore-mediated growth limitation of plant pathogens and biofilm formation, two processes relevant for plant growth-promoting activity.

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Complete Genome Sequence of the Naphthalene-Degrading Pseudomonas putida Strain ND6

Journal of Bacteriology ◽

10.1128/jb.01190-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 5154-5155 ◽

Cited By ~ 25

Author(s):

Shanshan Li ◽

Huabing Zhao ◽

Yaxiao Li ◽

Shumin Niu ◽

Baoli Cai

Keyword(s):

Pseudomonas Putida ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Content Type ◽

Degrading Bacterium ◽

Genes Encoding ◽

Chromosomal Sequence

ABSTRACTPseudomonas putidastrain ND6 is an efficient naphthalene-degrading bacterium. The complete genome of strain ND6 was sequenced and annotated. The genes encoding the enzymes involved in catechol degradation by theortho-cleavage pathway were found in the chromosomal sequence, which indicated that strain ND6 is able to metabolize naphthalene by the catecholmeta- andortho-cleavage pathways.

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Peptidoglycan-Associated Cyclic Lipopeptide Disrupts Viral Infectivity

Journal of Virology ◽

10.1128/jvi.01282-19 ◽

2019 ◽

Vol 93 (22) ◽

Cited By ~ 7

Author(s):

Bryan A. Johnson ◽

Adam Hage ◽

Birte Kalveram ◽

Megan Mears ◽

Jessica A. Plante ◽

...

Keyword(s):

Viral Infections ◽

Bacterial Species ◽

Enteric Viruses ◽

Hemorrhagic Fever ◽

Inhibitory Effect ◽

Microbial Interactions ◽

Enveloped Viruses ◽

Content Type ◽

Cyclic Lipopeptide

ABSTRACT Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from Bacillus subtilis reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection in vivo. Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection. IMPORTANCE In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.

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Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00781-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 9

Author(s):

Dongfei Han ◽

Ji-Young Ryu ◽

Robert A. Kanaly ◽

Hor-Gil Hur

Keyword(s):

Escherichia Coli ◽

Pseudomonas Putida ◽

Hypothetical Protein ◽

Aldehyde Group ◽

Agrobacterium Vitis ◽

T7 Promoter ◽

Content Type ◽

E Coli ◽

Cell Extracts ◽

Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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Genes Required for Aerial Growth, Cell Division, and Chromosome Segregation Are Targets of WhiA before Sporulation in Streptomyces venezuelae

mBio ◽

10.1128/mbio.00684-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 61

Author(s):

Matthew J. Bush ◽

Maureen J. Bibb ◽

Govind Chandra ◽

Kim C. Findlay ◽

Mark J. Buttner

Keyword(s):

Cell Division ◽

Chromosome Segregation ◽

Regulatory Networks ◽

Liquid Culture ◽

Biological Processes ◽

Streptomyces Venezuelae ◽

Content Type ◽

Master Regulators ◽

Aerial Growth ◽

Genes Encoding

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacteriumStreptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA inStreptomycesdevelopment and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus,Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed thatwhiAis required for the initiation of sporulation septation and chromosome segregation inS. venezuelae, and several genes encoding key proteins of theStreptomycescell division machinery, such asftsZ,ftsW, andftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiGand the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, andfilP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.IMPORTANCESince the initial identification of the genetic loci required forStreptomycesdevelopment, all of thebldandwhidevelopmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction ofStreptomyces venezuelaeas a new model system for the genus, a species that sporulates in liquid culture. Taking advantage ofS. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

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