Genetic and evolutionary characterization of the Major Facilitator Superfamily transporters of the antibacterial, Pantoea Natural Product 3

ABSTRACT Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter ofEscherichia coli and the glucose/galactose:H+symporter of Brucella abortus. In a wild-type B. subtilis genetic background, theglcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl α-glucoside uptake. In a Δptsgenetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a Δptsmutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl α-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.

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Identification and functional characterization of Penicillium marneffei major facilitator superfamily (MFS) transporters

Bioscience Biotechnology and Biochemistry ◽

10.1080/09168451.2020.1732185 ◽

2020 ◽

Vol 84 (7) ◽

pp. 1373-1383

Author(s):

Setyowati T. Utami ◽

Carissa I. Indriani ◽

Anom Bowolaksono ◽

Takashi Yaguchi ◽

Xinyue Chen ◽

...

Keyword(s):

Functional Characterization ◽

Major Facilitator Superfamily ◽

Penicillium Marneffei ◽

Major Facilitator ◽

Mfs Transporters

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ThemelREDCAOperon Encodes a Utilization System for the Raffinose Family of Oligosaccharides inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00109-19 ◽

2019 ◽

Vol 201 (15) ◽

Author(s):

Kambiz Morabbi Heravi ◽

Hildegard Watzlawick ◽

Josef Altenbuchner

Keyword(s):

Bacillus Subtilis ◽

Transcriptional Start Site ◽

Major Facilitator Superfamily ◽

Atp Binding ◽

Carbohydrate Utilization ◽

Content Type ◽

Storage Organs ◽

Major Facilitator ◽

Atp Binding Cassette

ABSTRACTBacillus subtilisis a heterotrophic soil bacterium that hydrolyzes different polysaccharides mainly found in the decomposed plants. These carbohydrates are mainly cellulose, hemicellulose, and the raffinose family of oligosaccharides (RFOs). RFOs are soluble α-galactosides, such as raffinose, stachyose, and verbascose, that rank second only after sucrose in abundance. Genome sequencing and transcriptome analysis ofB. subtilisindicated the presence of a putative α-galactosidase-encoding gene (melA) located in themsmRE-amyDC-melAoperon. Characterization of the MelA protein showed that it is a strictly Mn2+- and NAD+-dependent α-galactosidase able to hydrolyze melibiose, raffinose, and stachyose. Transcription of themsmER-amyDC-melAoperon is under control of a σA-type promoter located upstream ofmsmR(PmsmR), which is negatively regulated by MsmR. The activity of PmsmRwas induced in the presence of melibiose and raffinose. MsmR is a transcriptional repressor that binds to two binding sites at PmsmRlocated upstream of the −35 box and downstream of the transcriptional start site. MsmEX-AmyCD forms an ATP-binding cassette (ABC) transporter that probably transports melibiose into the cell. SincemsmRE-amyDC-melAis a melibiose utilization system, we renamed the operonmelREDCA.IMPORTANCEBacillus subtilisutilizes different polysaccharides produced by plants. These carbohydrates are primarily degraded by extracellular hydrolases, and the resulting oligo-, di-, and monosaccharides are transported into the cytosol via phosphoenolpyruvate-dependent phosphotransferase systems (PTS), major facilitator superfamily, and ATP-binding cassette (ABC) transporters. In this study, a new carbohydrate utilization system ofB. subtilisresponsible for the utilization of α-galactosides of the raffinose family of oligosaccharides (RFOs) was investigated. RFOs are synthesized from sucrose in plants and are mainly found in the storage organs of plant leaves. Our results revealed the modus operandi of a new carbohydrate utilization system inB. subtilis.

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Sialic acid acquisition in bacteria–one substrate, many transporters

Biochemical Society Transactions ◽

10.1042/bst20160056 ◽

2016 ◽

Vol 44 (3) ◽

pp. 760-765 ◽

Cited By ~ 16

Author(s):

Gavin H. Thomas

Keyword(s):

Sialic Acid ◽

Immune Evasion ◽

Sialic Acids ◽

Major Facilitator Superfamily ◽

Acid Uptake ◽

Gram Negative Bacteria ◽

Acetylneuraminic Acid ◽

Wide Range ◽

Major Facilitator

The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, β-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell.

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Characterization of Bacterial Drug Antiporters Homologous to Mammalian Neurotransmitter Transporters

Journal of Bacteriology ◽

10.1128/jb.187.21.7518-7525.2005 ◽

2005 ◽

Vol 187 (21) ◽

pp. 7518-7525 ◽

Cited By ~ 16

Author(s):

Eyal Vardy ◽

Sonia Steiner-Mordoch ◽

Shimon Schuldiner

Keyword(s):

Halobacterium Salinarum ◽

Major Facilitator Superfamily ◽

Control Measurement ◽

Multidrug Transporters ◽

Bacterial Proteins ◽

Distinct Cluster ◽

The Family ◽

Major Facilitator ◽

Dependent Transport

ABSTRACT Multidrug transporters are ubiquitous proteins, and, based on amino acid sequence similarities, they have been classified into several families. Here we characterize a cluster of archaeal and bacterial proteins from the major facilitator superfamily (MFS). One member of this family, the vesicular monoamine transporter (VMAT) was previously shown to remove both neurotransmitters and toxic compounds from the cytoplasm, thereby conferring resistance to their effects. A BLAST search of the available microbial genomes against the VMAT sequence yielded sequences of novel putative multidrug transporters. The new sequences along with VMAT form a distinct cluster within the dendrogram of the MFS, drug-proton antiporters. A comparison with other proteins in the family suggests the existence of a potential ion pair in the membrane domain. Three of these genes, from Mycobacterium smegmatis, Corynebacterium glutamicum, and Halobacterium salinarum, were cloned and functionally expressed in Escherichia coli. The proteins conferred resistance to fluoroquinolones and chloramphenicol (at concentrations two to four times greater than that of the control). Measurement of antibiotic accumulation in cells revealed proton motive force-dependent transport of those compounds.

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Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (thenrtPGene Product) from the CyanobacteriumNostoc punctiformeStrain ATCC 29133

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.60286 ◽

2006 ◽

Vol 70 (11) ◽

pp. 2682-2689 ◽

Cited By ~ 17

Author(s):

Makiko AICHI ◽

Saori YOSHIHARA ◽

Madoka YAMASHITA ◽

Shin-ichi MAEDA ◽

Kazuo NAGAI ◽

...

Keyword(s):

Major Facilitator Superfamily ◽

Major Facilitator

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Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus

Journal of Bacteriology ◽

10.1128/jb.185.23.6938-6949.2003 ◽

2003 ◽

Vol 185 (23) ◽

pp. 6938-6949 ◽

Cited By ~ 70

Author(s):

Tomotaka Tanabe ◽

Tatsuya Funahashi ◽

Hiroshi Nakao ◽

Shin-Ichi Miyoshi ◽

Sumio Shinoda ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

Major Facilitator Superfamily ◽

Homology Search ◽

Receptor Protein ◽

Pcr Analysis ◽

Insertion Mutation ◽

Outer Membrane Receptor ◽

Phenotype Analysis ◽

Major Facilitator

ABSTRACT In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, l-alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE, constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon, pvsC, encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE, encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA. Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

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