Contribution of Gene Loss to the Pathogenic Evolution of Burkholderia pseudomallei and Burkholderia mallei

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis. Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B. pseudomallei cannot. We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B. thailandensis. Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes. Analysis of the B. pseudomallei chromosome showed a deletion of the operon from this organism. This deletion was detected in all B. pseudomallei and Burkholderia mallei strains investigated. We cloned the B. thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B. pseudomallei 406e via homologous recombination. The resultant strain, B. pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source. Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e. Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose. These results suggest that the ability to metabolize l-arabinose reduces the virulence of B. pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes. The increase in virulence associated with the loss of these genes may have provided a selective advantage for B. pseudomallei as these organisms adapted to survival in animal hosts.

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Hidden resources in the Escherichia coli genome restore PLP synthesis and robust growth after deletion of the essential gene pdxB

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915569116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 24164-24173 ◽

Cited By ~ 4

Author(s):

Juhan Kim ◽

Jake J. Flood ◽

Michael R. Kristofich ◽

Cyrus Gidfar ◽

Andrew B. Morgenthaler ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Sole Carbon Source ◽

Essential Gene ◽

Alternative Pathway ◽

Gene Encoding ◽

Genes Encoding ◽

Phosphoglycerate Dehydrogenase ◽

Functional Pathway

PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5′-phosphate (PLP) in Escherichia coli. Surprisingly, incubation of the ∆pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes.

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A synthetic Calvin cycle enables autotrophic growth in yeast

10.1101/862599 ◽

2019 ◽

Author(s):

Thomas Gassler ◽

Michael Sauer ◽

Brigitte Gasser ◽

Diethard Mattanovich ◽

Matthias G. Steiger

Keyword(s):

Metabolic Engineering ◽

Carbon Source ◽

Sole Carbon Source ◽

Heterologous Protein ◽

Calvin Cycle ◽

Methylotrophic Yeast ◽

Genes Encoding ◽

Endogenous Genes ◽

Multiple Cell ◽

New Yeast

AbstractThe methylotrophic yeast Pichia pastoris is frequently used for heterologous protein production and it assimilates methanol efficiently via the xylulose-5-phosphate pathway. This pathway is entirely localized in the peroxisomes and has striking similarities to the Calvin-Benson-Bassham (CBB) cycle, which is used by a plethora of organisms like plants to assimilate CO2 and is likewise compartmentalized in chloroplasts. By metabolic engineering the methanol assimilation pathway of P. pastoris was re-wired to a CO2 fixation pathway resembling the CBB cycle. This new yeast strain efficiently assimilates CO2 into biomass and utilizes it as its sole carbon source, which changes the lifestyle from heterotrophic to autotrophic.In total eight genes, including genes encoding for RuBisCO and phosphoribulokinase, were integrated into the genome of P. pastoris, while three endogenous genes were deleted to block methanol assimilation. The enzymes necessary for the synthetic CBB cycle were targeted to the peroxisome. Methanol oxidation, which yields NADH, is employed for energy generation defining the lifestyle as chemoorganoautotrophic. This work demonstrates that the lifestyle of an organism can be changed from chemoorganoheterotrophic to chemoorganoautotrophic by metabolic engineering. The resulting strain can grow exponentially and perform multiple cell doublings on CO2 as sole carbon source with a µmax of 0.008 h−1.Graphical Abstract

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Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

Applied and Environmental Microbiology ◽

10.1128/aem.02059-15 ◽

2015 ◽

Vol 81 (24) ◽

pp. 8294-8306 ◽

Cited By ~ 16

Author(s):

Heba Khairy ◽

Jan Hendrik Wübbeler ◽

Alexander Steinbüchel

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Rhodococcus Erythropolis ◽

Model Organism ◽

Hypothetical Protein ◽

Gene Encoding ◽

Content Type ◽

Gene Coding ◽

Initial Cleavage ◽

Genes Encoding

ABSTRACTFourRhodococcusspp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. BecauseRhodococcus erythropolisstrain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis ofR. erythropolisMI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity ofnoxRandnox. The interruption mutant generated,R. erythropolisMI2noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently,noxwas overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

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Identification of genes encoding a novel ABC transporter in Lactobacillus delbrueckii for inulin polymers uptake

Scientific Reports ◽

10.1038/s41598-021-95356-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuji Tsujikawa ◽

Shu Ishikawa ◽

Iwao Sakane ◽

Ken-ichi Yoshida ◽

Ro Osawa

Keyword(s):

Bacillus Subtilis ◽

Carbon Source ◽

Heterologous Expression ◽

Gene Cluster ◽

Abc Transporter ◽

Transcriptome Analysis ◽

Sole Carbon Source ◽

Cultured Cells ◽

Lactobacillus Delbrueckii ◽

Genes Encoding

AbstractLactobacillus delbrueckii JCM 1002T grows on highly polymerized inulin-type fructans as its sole carbon source. When it was grown on inulin, a > 10 kb long gene cluster inuABCDEF (Ldb1381-1386) encoding a plausible ABC transporter was suggested to be induced, since a transcriptome analysis revealed that the fourth gene inuD (Ldb1384) was up-regulated most prominently. Although Bacillus subtilis 168 is originally unable to utilize inulin, it became to grow on inulin upon heterologous expression of inuABCDEF. When freshly cultured cells of the recombinant B. subtilis were then densely suspended in buffer containing inulin polymers and incubated, inulin gradually disappeared from the buffer and accumulated in the cells without being degraded, whereas levan-type fructans did not disappear. The results imply that inuABCDEF might encode a novel ABC transporter in L. delbrueckii to “monopolize” inulin polymers selectively, thereby, providing a possible advantage in competition with other concomitant inulin-utilizing bacteria.

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Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei

Infection and Immunity ◽

10.1128/iai.00102-17 ◽

2017 ◽

Vol 85 (8) ◽

Cited By ~ 11

Author(s):

Shawn M. Zimmerman ◽

Jeremy S. Dyke ◽

Tomislav P. Jelesijevic ◽

Frank Michel ◽

Eric R. Lafontaine ◽

...

Keyword(s):

Mutant Strain ◽

Protective Immunity ◽

Burkholderia Pseudomallei ◽

Lipolytic Activity ◽

Burkholderia Mallei ◽

Wild Type ◽

Content Type ◽

Related Organism ◽

Genes Encoding

ABSTRACT Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei. Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo. Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery.

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Bioconversion of biphenyl to a polyhydroxyalkanoate copolymer by Alcaligenes denitrificans A41

10.21203/rs.3.rs-36822/v2 ◽

2020 ◽

Author(s):

Taito Yajima ◽

Mizuki Nagatomo ◽

Aiko Wakabayashi ◽

Michio Sato ◽

Seiichi Taguchi ◽

...

Keyword(s):

Molecular Weight ◽

Carbon Source ◽

Sole Carbon Source ◽

Average Molecular Weight ◽

Gel Permeation Chromatography ◽

Nitrogen Sources ◽

Dry Weight ◽

Environmental Pollutant ◽

Genes Encoding ◽

Copolymer Poly

Abstract A polyhydroxyalkanoate (PHA) copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)], was biosynthesized from biphenyl as the sole carbon source using Alcaligenes (currently Achromobacter) denitrificans A41. This strain is capable of degrading polychlorinated biphenyls (PCBs) and biphenyl. This proof-of-concept of the conversion of aromatic chemicals such as the environmental pollutant PCBs/biphenyl to eco-friendly products such as biodegradable polyester PHA was inspired by the uncovering of two genes encoding PHA synthases in the A. denitrificans A41 genome. When the carbon/nitrogen (C/N) ratio was set at 21, the cellular P(3HB-co-3HV) content in strain A41 reached its highest value of 10.1% of the cell dry weight (CDW). A two-step cultivation protocol improved the accumulation of P(3HB-co-3HV) by up to 26.2% of the CDW, consisting of 13.0 mol% 3HV when grown on minimum salt medium without nitrogen sources. The highest cellular content of P(3HB-co-3HV) (47.6% of the CDW) was obtained through the two-step cultivation of strain A41 on biphenyl as the sole carbon source. The purified copolymer had ultra-high molecular weight (weight-average molecular weight of 3.5 × 106), as revealed through gel-permeation chromatography. Based on the genomic information related to both polymer synthesis and biphenyl degradation, we finally proposed a metabolic pathway for the production of P(3HB-co-3HV) associated with the degradation of biphenyl by strain A41.

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In VitroandIn VivoStudies of Monoclonal Antibodies with Prominent Bactericidal Activity against Burkholderia pseudomallei and Burkholderia mallei

Clinical and Vaccine Immunology ◽

10.1128/cvi.00533-10 ◽

2011 ◽

Vol 18 (5) ◽

pp. 825-834 ◽

Cited By ~ 32

Author(s):

Shimin Zhang ◽

Shaw-Huey Feng ◽

Bingjie Li ◽

Hyung-Yong Kim ◽

Joe Rodriguez ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Bactericidal Activity ◽

Burkholderia Pseudomallei ◽

Protective Efficacy ◽

Lethal Dose ◽

Burkholderia Mallei ◽

Content Type ◽

Bactericidal Activities

ABSTRACTOur laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) againstBurkholderia pseudomalleiandBurkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by anin vitroopsonic assay. Then, thein vivoprotective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities againstB. pseudomalleiand/orB. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Ourin vivostudy showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of ourin vitrostudy. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of theBurkholderiabacteria.

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Transport and Catabolism of Carbohydrates by Neisseria meningitidis

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000447093 ◽

2016 ◽

Vol 26 (5) ◽

pp. 320-332 ◽

Cited By ~ 4

Author(s):

Meriem Derkaoui ◽

Ana Antunes ◽

Jamila Nait Abdallah ◽

Sandrine Poncet ◽

Alain Mazé ◽

...

Keyword(s):

Carbon Source ◽

Transgenic Mice ◽

Neisseria Meningitidis ◽

Pentose Phosphate Pathway ◽

Sole Carbon Source ◽

Minimal Medium ◽

Pentose Phosphate ◽

Key Enzymes ◽

The Real ◽

Genes Encoding

We identified the genes encoding the proteins for the transport of glucose and maltose in Neisseria meningitidis strain 2C4-3. A mutant deleted for NMV_1892(glcP) no longer grew on glucose and deletion of NMV_0424(malY) prevented the utilization of maltose. We also purified and characterized glucokinase and α-phosphoglucomutase, which catalyze early catabolic steps of the two carbohydrates. N. meningitidis catabolizes the two carbohydrates either via the Entner-Doudoroff (ED) pathway or the pentose phosphate pathway, thereby forming glyceraldehyde-3-P and either pyruvate or fructose-6-P, respectively. We purified and characterized several key enzymes of the two pathways. The genes required for the transformation of glucose into gluconate-6-P and its further catabolism via the ED pathway are organized in two adjacent operons. N. meningitidis also contains genes encoding proteins which exhibit similarity to the gluconate transporter (NMV_2230) and gluconate kinase (NMV_2231) of Enterobacteriaceae and Firmicutes. However, gluconate might not be the real substrate of NMV_2230 because N. meningitidis was not able to grow on gluconate as the sole carbon source. Surprisingly, deletion of NMV_2230 stimulated growth in minimal medium in the presence and absence of glucose and drastically slowed the clearance of N. meningitidis cells from transgenic mice after intraperitoneal challenge.

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Transformation of Isopropylamine to l-Alaninol by Pseudomonas sp. Strain KIE171 Involves N-Glutamylated Intermediates

Applied and Environmental Microbiology ◽

10.1128/aem.68.5.2368-2375.2002 ◽

2002 ◽

Vol 68 (5) ◽

pp. 2368-2375 ◽

Cited By ~ 21

Author(s):

Susana I. de Azevedo Wäsch ◽

Jan R. van der Ploeg ◽

Tere Maire ◽

Alice Lebreton ◽

Andreas Kiener ◽

...

Keyword(s):

Enzymatic Hydrolysis ◽

Carbon Source ◽

Alcohol Dehydrogenase ◽

Sole Carbon Source ◽

The Other ◽

Pseudomonas Sp ◽

Component System ◽

Aldehyde Dehydrogenases ◽

Transposon Insertion ◽

Do So

ABSTRACT Pseudomonas sp. strain KIE171 was able to grow with isopropylamine or l-alaninol [S-(+)-2-amino-1-propanol] as the sole carbon source, but not with d-alaninol. To investigate the hypothesis that l-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and l-alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to l-alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced. Two clusters, one comprising eight ipu (isopropylamine utilization) genes (ipuABCDEFGH) and the other encompassing two genes (ipuI and orf259), were identified. Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG. The next step, the formation of γ-glutamyl-isopropylamide from isopropylamine, ATP, and l-glutamate, was shown to be catalyzed by IpuC, a γ-glutamylamide synthetase. γ-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding γ-glutamyl-l-alaninol [γ(l-glutamyl)-l-hydroxy-isopropylamide]. Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate l-alaninol and to regenerate l-glutamate. No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters. Presumably, l-alaninol is oxidized by an alcohol dehydrogenase to yield l-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde. Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either l-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.

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Similarities between the antABC-Encoded Anthranilate Dioxygenase and the benABC-Encoded Benzoate Dioxygenase of Acinetobacter sp. Strain ADP1

Journal of Bacteriology ◽

10.1128/jb.180.17.4466-4474.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4466-4474 ◽

Cited By ~ 56

Author(s):

Becky M. Bundy ◽

Alan L. Campbell ◽

Ellen L. Neidle

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Gene Clusters ◽

Open Reading Frames ◽

Wild Type ◽

Aromatic Compound Degradation ◽

Genes Encoding ◽

Dna Fragment ◽

Benzoate Dioxygenase ◽

Reading Frames

ABSTRACT Acinetobacter sp. strain ADP1 can use benzoate or anthranilate as a sole carbon source. These structurally similar compounds are independently converted to catechol, allowing further degradation to proceed via the β-ketoadipate pathway. In this study, the first step in anthranilate catabolism was characterized. A mutant unable to grow on anthranilate, ACN26, was selected. The sequence of a wild-type DNA fragment that restored growth revealed theantABC genes, encoding 54-, 19-, and 39-kDa proteins, respectively. The deduced AntABC sequences were homologous to those of class IB multicomponent aromatic ring-dihydroxylating enzymes, including the dioxygenase that initiates benzoate catabolism. Expression of antABC in Escherichia coli, a bacterium that normally does not degrade anthranilate, enabled the conversion of anthranilate to catechol. Unlike benzoate dioxygenase (BenABC), anthranilate dioxygenase (AntABC) catalyzed catechol formation without requiring a dehydrogenase. In Acinetobacter mutants,benC substituted for antC during growth on anthranilate, suggesting relatively broad substrate specificity of the BenC reductase, which transfers electrons from NADH to the terminal oxygenase. In contrast, the benAB genes did not substitute for antAB. An antA point mutation in ACN26 prevented anthranilate degradation, and this mutation was independent of a mucK mutation in the same strain that prevented exogenous muconate degradation. Anthranilate induced expression of antA, although no associated transcriptional regulators were identified. Disruption of three open reading frames in the immediate vicinity ofantABC did not prevent the use of anthranilate as a sole carbon source. TheantABC genes were mapped on the ADP1 chromosome and were not linked to the two known supraoperonic gene clusters involved in aromatic compound degradation.

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