scholarly journals Impact of Vitamin B12on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

2012 ◽  
Vol 78 (22) ◽  
pp. 8025-8032 ◽  
Author(s):  
Anika Reinhold ◽  
Martin Westermann ◽  
Jana Seifert ◽  
Martin von Bergen ◽  
Torsten Schubert ◽  
...  
Keyword(s):  
Gene Cluster ◽  
De Novo ◽  
Anaerobic Bacteria ◽  
Vitamin B ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Gene Level ◽  
Desulfitobacterium Hafniense ◽  
Pce Dehalogenase ◽  
The Impact

ABSTRACTCorrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable ofde novocorrinoid biosynthesis (e.g.,Desulfitobacteriumspp.) or dependent on exogenous vitamin B12(e.g.,Dehalococcoidesspp.). In this study, the impact of exogenous vitamin B12(cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positiveDesulfitobacterium hafniensestrain Y51, a bacterium able to synthesize corrinoidsde novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of thepcegene cluster. In the absence of vitamin B12, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B12, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in thepceAgene level, indicating that exogenous vitamin B12hampered the transposition of thepcegene cluster. In the absence or presence of exogenous vitamin B12, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B12is not incorporated into the PceA precursor, even though it affects the transposition of thepcegene cluster.

scholarly journals Thermotoga lettingae Can Salvage Cobinamide To Synthesize Vitamin B12

2013 ◽  
Vol 79 (22) ◽  
pp. 7006-7012 ◽  
Author(s):  
Nicholas C. Butzin ◽  
Michael A. Secinaro ◽  
Kristen S. Swithers ◽  
J. Peter Gogarten ◽  
Kenneth M. Noll
Keyword(s):  
Gene Cluster ◽  
Common Ancestor ◽  
De Novo ◽  
Recent Common Ancestor ◽  
Vitamin B ◽  
Ancestral State ◽  
Salvage Pathway ◽  
Content Type ◽  
Most Recent Common Ancestor

ABSTRACTWe recently reported that theThermotogalesacquired the ability to synthesize vitamin B12by acquisition of genes from two distantly related lineages,ArchaeaandFirmicutes(K. S. Swithers et al., Genome Biol. Evol. 4:730–739, 2012). Ancestral state reconstruction suggested that the cobinamide salvage gene cluster was present in theThermotogales' most recent common ancestor. We also predicted thatThermotoga lettingaecould not synthesize B12de novobut could use the cobinamide salvage pathway to synthesize B12. In this study, these hypotheses were tested, and we found thatTt. lettingaedid not synthesize B12de novobut salvaged cobinamide. The growth rate ofTt. lettingaeincreased with the addition of B12or cobinamide to its medium. It synthesized B12when the medium was supplemented with cobinamide, and no B12was detected in cells grown on cobinamide-deficient medium. Upstream of the cobinamide salvage genes is a putative B12riboswitch. In other organisms, B12riboswitches allow for higher transcriptional activity in the absence of B12. WhenTt. lettingaewas grown with no B12, the salvage genes were upregulated compared to cells grown with B12or cobinamide. Another gene cluster with a putative B12riboswitch upstream is thebtuFCDABC transporter, and it showed a transcription pattern similar to that of the cobinamide salvage genes. The BtuF proteins from species that can and cannot salvage cobinamides were shownin vitroto bind both B12and cobinamide. These results suggest thatThermotogalesspecies can use the BtuFCD transporter to import both B12and cobinamide, even if they cannot salvage cobinamide.

scholarly journals Unexpected Specificity of Interspecies Cobamide Transfer from Geobacter spp. to Organohalide-Respiring Dehalococcoides mccartyi Strains

2012 ◽  
Vol 78 (18) ◽  
pp. 6630-6636 ◽  
Author(s):  
Jun Yan ◽  
Kirsti M. Ritalahti ◽  
Darlene D. Wagner ◽  
Frank E. Löffler
Keyword(s):  
Reductive Dechlorination ◽  
De Novo ◽  
Synthetic Medium ◽  
Geobacter Sulfurreducens ◽  
Rrna Gene ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Mccartyi ◽  
Gene Copies ◽  
Pure Cultures

ABSTRACTDehalococcoides mccartyistrains conserve energy from reductive dechlorination reactions catalyzed by corrinoid-dependent reductive dehalogenase enzyme systems.Dehalococcoideslacks the ability forde novocorrinoid synthesis, and pure cultures require the addition of cyanocobalamin (vitamin B12) for growth. In contrast,Geobacter lovleyi, which dechlorinates tetrachloroethene tocis-1,2-dichloroethene (cis-DCE), and the nondechlorinating speciesGeobacter sulfurreducenshave complete sets of cobamide biosynthesis genes and produced 12.9 ± 2.4 and 24.2 ± 5.8 ng of extracellular cobamide per liter of culture suspension, respectively, during growth with acetate and fumarate in a completely synthetic medium.G. lovleyi-D. mccartyistrain BAV1 or strain FL2 cocultures provided evidence for interspecies corrinoid transfer, andcis-DCE was dechlorinated to vinyl chloride and ethene concomitant withDehalococcoidesgrowth. In contrast, negligible increase inDehalococcoides16S rRNA gene copies and insignificant dechlorination occurred inG. sulfurreducens-D. mccartyistrain BAV1 or strain FL2 cocultures. Apparently,G. lovleyiproduces a cobamide that complementsDehalococcoides' nutritional requirements, whereasG. sulfurreducensdoes not. Interestingly,Dehalococcoidesdechlorination activity and growth could be restored inG. sulfurreducens-Dehalococcoidescocultures by adding 10 μM 5′,6′-dimethylbenzimidazole. Observations made with theG. sulfurreducens-Dehalococcoidescocultures suggest that the exchange of the lower ligand generated a cobalamin, which supportedDehalococcoidesactivity. These findings have implications forin situbioremediation and suggest that the corrinoid metabolism ofDehalococcoidesmust be understood to faithfully predict, and possibly enhance, reductive dechlorination activities.

scholarly journals Inactivation of thyA in Staphylococcus aureus Attenuates Virulence and Has a Strong Impact on Metabolism and Virulence Gene Expression

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Andre Kriegeskorte ◽  
Desiree Block ◽  
Mike Drescher ◽  
Nadine Windmüller ◽  
Alexander Mellmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Thymidylate Synthase ◽  
Molecular Basis ◽  
De Novo ◽  
Strong Impact ◽  
Small Colony Variants ◽  
Content Type ◽  
Long Term Treatment ◽  
Small Colony ◽  
The Impact

ABSTRACTStaphylococcus aureusthymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronicS. aureusinfections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS;thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations inthyAwere leading to inactivity of TS proteins, and TS inactivity led to tremendous impact onS. aureusphysiology and virulence. Whole DNA microarray analysis of the constructed ΔthyAmutant identified severe alterations compared to the wild type. Important virulence regulators (agr,arlRS,sarA) and major virulence determinants (hla,hlb,sspAB, andgeh) were downregulated, while genes important for colonization (fnbA,fnbB,spa,clfB,sdrC, andsdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyAmutant was strongly attenuated in virulence models, including aCaenorhabditis eleganskilling model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed thatthyAactivity has a major role forS. aureusvirulence and physiology.IMPORTANCEThymidine-dependent small-colony variants (TD-SCVs) ofStaphylococcus aureuscarry mutations in the thymidylate synthase (TS) gene (thyA) responsible forde novosynthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistantS. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact onS. aureusvirulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

scholarly journals Effects of Chloromethanes on Growth of and Deletion of the pce Gene Cluster in Dehalorespiring Desulfitobacterium hafniense Strain Y51

2006 ◽  
Vol 72 (9) ◽  
pp. 5998-6003 ◽  
Author(s):  
Taiki Futagami ◽  
Takehito Yamaguchi ◽  
Shun-ichi Nakayama ◽  
Masatoshi Goto ◽  
Kensuke Furukawa
Keyword(s):  
Carbon Tetrachloride ◽  
Growth Inhibition ◽  
Gene Cluster ◽  
Inhibitory Effect ◽  
Large Deletion ◽  
Growth Inhibitory Effect ◽  
Reductive Dehalogenase ◽  
Growth Inhibitory ◽  
Desulfitobacterium Hafniense ◽  
Significant Growth Inhibition

ABSTRACT The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 μM, but not carbon dichloride even at 1 mM.

scholarly journals Flexible Cobamide Metabolism in Clostridioides (Clostridium) difficile 630 Δerm

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Amanda N. Shelton ◽  
Xun Lyu ◽  
Michiko E. Taga
Keyword(s):  
De Novo ◽  
Aminolevulinic Acid ◽  
Genomic Analysis ◽  
Opportunistic Pathogen ◽  
Vitamin B ◽  
Uptake System ◽  
Human Gut ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent pathways encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro. Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinic acid or the late intermediate cobinamide (Cbi) and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in the btuFCD loci of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile. IMPORTANCE The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, although it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo but produces them when given cobamide precursors. A better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.

scholarly journals Quantitative Analysis of the Relative Transcript Levels of Four Chlorophenol Reductive Dehalogenase Genes in Desulfitobacterium hafniense PCP-1 Exposed to Chlorophenols

2011 ◽  
Vol 77 (17) ◽  
pp. 6261-6264 ◽  
Author(s):  
Ariane Bisaillon ◽  
Réjean Beaudet ◽  
François Lépine ◽  
Richard Villemur
Keyword(s):  
Quantitative Analysis ◽  
Transcript Levels ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Reductive Dehalogenase Genes ◽  
Desulfitobacterium Hafniense

ABSTRACTRelative to those of unexposed cultures, the transcript levels of the four CprA-type reductive dehalogenase genes (cprA2,cprA3,cprA4, andcprA5) inDesulfitobacterium hafniensePCP-1 were measured in cultures exposed to chlorophenols. In 2,4,6-trichlorophenol-amended cultures,cprA2andcprA3were upregulated, as wascprA5, but concomitantly with the appearance of 2,4-dichlorophenol (DCP). In 3,5-DCP-amended cultures, onlycprA5was upregulated. In pentachlorophenol-amended cultures grown for 12 h,cprA2andcprA3were upregulated but notcprA5. cprA4was not upregulated significantly in cultures containing any tested chlorophenols.

scholarly journals Folate rescues vitamin B12 depletion-induced inhibition of nuclear thymidylate biosynthesis and genome instability

2017 ◽  
Vol 114 (20) ◽  
pp. E4095-E4102 ◽  
Author(s):  
Ashley M. Palmer ◽  
Elena Kamynina ◽  
Martha S. Field ◽  
Patrick J. Stover
Keyword(s):  
De Novo ◽  
Genome Instability ◽  
Megaloblastic Anemia ◽  
Tight Binding ◽  
Vitamin B ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Histone H2ax ◽  
Inhibition Of Dna Synthesis ◽  
The Impact

Clinical vitamin B12 deficiency can result in megaloblastic anemia, which results from the inhibition of DNA synthesis by trapping folate cofactors in the form of 5-methyltetrahydrofolate (5-methylTHF) and subsequent inhibition of de novo thymidylate (dTMP) biosynthesis. In the cytosol, vitamin B12 functions in the remethylation of homocysteine to methionine, which regenerates THF from 5-methylTHF. In the nucleus, THF is required for de novo dTMP biosynthesis, but it is not understood how 5-methylTHF accumulation in the cytosol impairs nuclear dTMP biosynthesis. The impact of vitamin B12 depletion on nuclear de novo dTMP biosynthesis was investigated in methionine synthase-null human fibroblast and nitrous oxide-treated HeLa cell models. The nucleus was the most sensitive cellular compartment to 5-methylTHF accumulation, with levels increasing greater than fourfold. Vitamin B12 depletion decreased de novo dTMP biosynthesis capacity by 5–35%, whereas de novo purine synthesis, which occurs in the cytosol, was not affected. Phosphorylated histone H2AX (γH2AX), a marker of DNA double-strand breaks, was increased in vitamin B12 depletion, and this effect was exacerbated by folate depletion. These studies also revealed that 5-formylTHF, a slow, tight-binding inhibitor of serine hydroxymethyltransferase (SHMT), was enriched in nuclei, accounting for 35% of folate cofactors, explaining previous observations that nuclear SHMT is not a robust source of one-carbons for de novo dTMP biosynthesis. These findings indicate that a nuclear 5-methylTHF trap occurs in vitamin B12 depletion, which suppresses de novo dTMP biosynthesis and causes DNA damage, accounting for the pathophysiology of megaloblastic anemia observed in vitamin B12 and folate deficiency.

scholarly journals Identification of a Novel Cobamide Remodeling Enzyme in the Beneficial Human Gut Bacterium Akkermansia muciniphila

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Kenny C. Mok ◽  
Olga M. Sokolovskaya ◽  
Alexa M. Nicolas ◽  
Zachary F. Hallberg ◽  
Adam Deutschbauer ◽  
...  
Keyword(s):  
De Novo ◽  
Structural Diversity ◽  
Vitamin B ◽  
Human Gut ◽  
Diverse Range ◽  
Content Type ◽  
Akkermansia Muciniphila ◽  
Binding Domains ◽  
Domains Of Life ◽  
Lower Ligand

ABSTRACT The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain MucT is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila MucT is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila MucT were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling—the removal and replacement of the lower ligand—despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila. We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli. Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment. IMPORTANCE Cobamides, comprising the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discovered that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via a process termed cobamide remodeling. We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications for understanding the ecological role of A. muciniphila in the gut and the functions of other bacteria that produce this enzyme.

scholarly journals Impact of Facultative Bacteria on the Metabolic Function of an Obligate Insect-Bacterial Symbiosis

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Frances Blow ◽  
Nana Y. D. Ankrah ◽  
Noah Clark ◽  
Imhoi Koo ◽  
Erik L. Allman ◽  
...  
Keyword(s):  
Acyrthosiphon Pisum ◽  
De Novo ◽  
Microbial Colonization ◽  
Content Type ◽  
Animal Host ◽  
Obligate Intracellular ◽  
Beneficial Microorganisms ◽  
Metabolic Interactions ◽  
Host Metabolism ◽  
The Impact

ABSTRACT Beneficial microorganisms associated with animals derive their nutritional requirements entirely from the animal host, but the impact of these microorganisms on host metabolism is largely unknown. The focus of this study was the experimentally tractable tripartite symbiosis between the pea aphid Acyrthosiphon pisum, its obligate intracellular bacterial symbiont Buchnera, and the facultative bacterium Hamiltonella which is localized primarily to the aphid hemolymph (blood). Metabolome experiments on, first, multiple aphid genotypes that naturally bear or lack Hamiltonella and, second, one aphid genotype from which Hamiltonella was experimentally eliminated revealed no significant effects of Hamiltonella on aphid metabolite profiles, indicating that Hamiltonella does not cause major reconfiguration of host metabolism. However, the titer of just one metabolite, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), displayed near-significant enrichment in Hamiltonella-positive aphids in both metabolome experiments. AICAR is a by-product of biosynthesis of the essential amino acid histidine in Buchnera and, hence, an index of histidine biosynthetic rates, suggesting that Buchnera-mediated histidine production is elevated in Hamiltonella-bearing aphids. Consistent with this prediction, aphids fed on [13C]histidine yielded a significantly elevated 12C/13C ratio of histidine in Hamiltonella-bearing aphids, indicative of increased (∼25%) histidine synthesized de novo by Buchnera. However, in silico analysis predicted an increase of only 0.8% in Buchnera histidine synthesis in Hamiltonella-bearing aphids. We hypothesize that Hamiltonella imposes increased host demand for histidine, possibly for heightened immune-related functions. These results demonstrate that facultative bacteria can alter the dynamics of host metabolic interactions with co-occurring microorganisms, even when the overall metabolic homeostasis of the host is not substantially perturbed. IMPORTANCE Although microbial colonization of the internal tissues of animals generally causes septicemia and death, various animals are persistently associated with benign or beneficial microorganisms in their blood or internal organs. The metabolic consequences of these persistent associations for the animal host are largely unknown. Our research on the facultative bacterium Hamiltonella, localized primarily to the hemolymph of pea aphids, demonstrated that although Hamiltonella imposed no major reconfiguration of the aphid metabolome, it did alter the metabolic relations between the aphid and its obligate intracellular symbiont, Buchnera. Specifically, Buchnera produced more histidine in Hamiltonella-positive aphids to support both Hamiltonella demand for histidine and Hamiltonella-induced increase in host demand. This study demonstrates how microorganisms associated with internal tissues of animals can influence specific aspects of metabolic interactions between the animal host and co-occurring microorganisms.

scholarly journals Involvement of Vitamin B6Biosynthesis Pathways in the Insecticidal Activity of Photorhabdus luminescens

2016 ◽  
Vol 82 (12) ◽  
pp. 3546-3553 ◽  
Author(s):  
Kazuki Sato ◽  
Toyoshi Yoshiga ◽  
Koichi Hasegawa
Keyword(s):  
Insecticidal Activity ◽  
Genetic Screening ◽  
De Novo ◽  
Entomopathogenic Nematode ◽  
Vitamin B ◽  
Photorhabdus Luminescens ◽  
Content Type ◽  
C Elegans ◽  
Growth Deficiency ◽  
Highly Virulent

ABSTRACTPhotorhabdus luminescensis a Gram-negative entomopathogenic bacterium which symbiotically associates with the entomopathogenic nematodeHeterorhabditis bacteriophora.P. luminescensis highly virulent to many insects and nonsymbiotic nematodes, includingCaenorhabditis elegans. To understand the virulence mechanisms ofP. luminescens, we obtained virulence-deficient and -attenuated mutants againstC. elegansthrough a transposon-mutagenized library. From the genetic screening, we identified thepdxBgene, encoding erythronate-4-phosphate dehydrogenase, as required forde novovitamin B6biosynthesis. Mutation inpdxBcaused growth deficiency ofP. luminescensin nutrient-poor medium, which was restored under nutrient-rich conditions or by supplementation with pyridoxal 5′-phosphate (PLP), an active form of vitamin B6. Supplementation with three other B6vitamers (pyridoxal, pyridoxine, and pyridoxamine) also restored the growth of thepdxBmutant, suggesting the existence of a salvage pathway for vitamin B6biosynthesis inP. luminescens. Moreover, supplementation with PLP restored the virulence-deficient phenotype againstC. elegans. Combining these results with the fact thatpdxBmutation also caused attenuation of insecticidal activity, we concluded that the production of appropriate amounts of vitamin B6is critical forP. luminescenspathogenicity.IMPORTANCEThe Gram-negative entomopathogenic bacteriumPhotorhabdus luminescenssymbiotically associates with the entomopathogenic nematodeHeterorhabditis bacteriophora.P. luminescensis highly virulent to many insects and nonsymbiotic nematodes, includingCaenorhabditis elegans. We have obtained several virulence-deficient and -attenuatedP. luminescensmutants againstC. elegansthrough genetic screening. From the genetic analysis, we present the vitamin B6biosynthetic pathways inP. luminescensthat are important for its insecticidal activity. Mutation inpdxB, encoding erythronate-4-phosphate dehydrogenase and required for thede novovitamin B6biosynthesis pathway, caused virulence deficiency againstC. elegansand growth deficiency ofP. luminescensin nutrient-poor medium. Because such phenotypes were restored under nutrient-rich conditions or by supplementation with B6vitamers, we showed the presence of the two vitamin B6synthetic pathways (de novoand salvage) inP. luminescensand also showed that the ability to produce an appropriate amount of vitamin B6is critical forP. luminescenspathogenicity.

Sign in / Sign up

Export Citation Format

Share Document