Functional Characterization of Transporters for L-Aspartate in Bacillus licheniformis

Amino acid efflux and influx transport systems play vital roles in industrial microorganisms’ cell growth and metabolism. However, although biochemically characterized, most amino acid transporters remain unknown at the molecular level in Bacillus licheniformis. This study focuses on the molecular and functional characterizations of three transporters, YdgF, YvbW, and YveA, mainly when catalyzing the cross-membrane flux of L-Aspartate. When growing in the minimal medium with L-Asp as the only carbon and nitrogen source, the growth of strains lacking proteins YdgF, YvbW, and YveA was significantly inhibited compared with wild-type strains, while supplementing the expression of the corresponding proteins in the single-gene knockout strains can alleviate the inhibition to some extent. Upon overexpression, the recombinant proteins mediate the accumulation of L-aspartate to varying degrees. Compared with wild-type strains, the single knockout strains of the three protein genes exhibited reduced absorption of L-aspartate. In addition, this paper focuses on the effects of these three proteins on the absorption of β-alanine, L-glutamate, D-serine, D-alanine, and glycine.

Download Full-text

Functional Characterization of Transporters for L-Aspartate in Bacillus licheniformis

Fermentation ◽

10.3390/fermentation8010022 ◽

2022 ◽

Vol 8 (1) ◽

pp. 22

Author(s):

Hanrong Wang ◽

Youran Li ◽

Fengxu Xiao ◽

Yupeng Zhang ◽

Guiyang Shi ◽

...

Keyword(s):

Bacillus Licheniformis ◽

Gene Knockout ◽

Single Gene ◽

Functional Characterization ◽

Transport Systems ◽

Wild Type ◽

Carbon And Nitrogen ◽

Influx Transport ◽

Amino Acid Efflux ◽

Type Strains

Amino acid efflux and influx transport systems play vital roles in industrial microorganisms’ cell growth and metabolism. However, although biochemically characterized, most of them remain unknown at the molecular level in Bacillus licheniformis. In this study, three proteins, namely, YdgF, YvbW, and YveA, were predicted to be involved in the active transport of L-aspartate (L-Asp). This was verified by manipulating their encoding genes. When growing in the minimal medium with L-Asp as the only carbon and nitrogen source, the growth of strains lacking proteins YdgF, YvbW, and YveA was significantly inhibited compared with the wild-type strains, while supplementing the expression of the corresponding proteins in the single-gene knockout strains could alleviate the inhibition. Upon overexpression, the recombinant proteins mediated the accumulation of L-aspartate to varying degrees. Compared with the wild-type strains, the single knockout strains of the three protein genes exhibited reduced absorption of L-aspartate. In addition, this study focused on the effects of these three proteins on the absorption of β-alanine, L-glutamate, D-serine, D-alanine, and glycine.

Download Full-text

Placental amino acid transport

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.6.c1321 ◽

1995 ◽

Vol 268 (6) ◽

pp. C1321-C1331 ◽

Cited By ~ 85

Author(s):

A. J. Moe

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Human Placenta ◽

Amino Acid Transport ◽

Single Layer ◽

Maternal Blood ◽

Transport Systems ◽

Amino Acid Transporters ◽

Acid Transport ◽

Principal Mechanism

Normal fetal growth and development depend on a continuous supply of amino acids from the mother to the fetus. The placenta is responsible for the transfer of amino acids between the two circulations. The human placenta is hemomonochorial, meaning that the maternal and fetal circulations are separated by a single layer of polarized epithelium called the syncytiotrophoblast, which is in direct contact with maternal blood. Transport proteins located in the microvillous and basal membranes of the syncytiotrophoblast are the principal mechanism for transfer from maternal blood to fetal blood. Knowledge of the function and regulation of syncytiotrophoblast amino acid transporters is of great importance in understanding the mechanism of placental transport and potentially improving fetal and newborn outcomes. The development of methods for the isolation of microvillous and basal membrane vesicles from human placenta over the past two decades has contributed greatly to this understanding. Now a primary cultured trophoblast model is available to study amino acid transport and regulation as the cells differentiate. The types of amino acid transporters and their distribution between the syncytiotrophoblast microvillous and basal membranes are somewhat unique compared with other polarized epithelia. These differences may reflect the unusual circumstance of this epithelium that is exposed to blood on both sides. The current state of knowledge as to the types of transport systems present in syncytiotrophoblast, their regulation, and the effects of maternal consumption of drugs on transport are discussed.

Download Full-text

Isolation and Characterization of vicH, Encoding a New Pleiotropic Regulator in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.182.7.2026-2032.2000 ◽

2000 ◽

Vol 182 (7) ◽

pp. 2026-2032 ◽

Cited By ~ 29

Author(s):

Christian Tendeng ◽

Cyril Badaut ◽

Evelyne Krin ◽

Pierre Gounon ◽

Saravuth Ngo ◽

...

Keyword(s):

Amino Acid ◽

Vibrio Cholerae ◽

Genomic Library ◽

Single Gene ◽

Wild Type ◽

Semisolid Medium ◽

E Coli ◽

Isolation And Characterization ◽

Serovar Typhimurium

ABSTRACT During the last decade, the hns gene and its product, the H-NS protein, have been extensively studied in Escherichia coli. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in E. coli and inSalmonella enterica serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of vicH, an hns-like gene in Vibrio cholerae, the etiological agent of cholera. This gene was isolated from a V. cholerae genomic library by complementation of different phenotypes associated with anhns mutation in E. coli. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in E. coli. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. ThevicH gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A V. choleraewild-type strain expressing a vicHΔ92 gene lacking its 3′ end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the vicH wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in V. cholerae, in particular by affecting flagellum biosynthesis.

Download Full-text

Functional characterization of twoS-nitroso-l-cysteine transporters, which mediate movement of NO equivalents into vascular cells

AJP Cell Physiology ◽

10.1152/ajpcell.00382.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. C1263-C1271 ◽

Cited By ~ 28

Author(s):

Sheng Li ◽

A. Richard Whorton

Keyword(s):

Smooth Muscle ◽

Amino Acid ◽

Smooth Muscle Cells ◽

Functional Characterization ◽

Muscle Cells ◽

Cellular Responses ◽

Functional System ◽

Xenopus Laevis Oocytes ◽

Amino Acid Transporters ◽

System L

System L amino acid transporters have been shown to be responsible for cellular uptake of S-nitroso-l-cysteine (l-CSNO). In this study, we examined the characteristics of l-CSNO uptake in Xenopus laevis oocytes expressing system L transporters and found that uptake increased only when both 4F2 heavy chain (4F2HC) and either L-type amino acid transporter 1 (LAT1) or LAT2 light chain were coexpressed. The Kmfor transport was 57 ± 8 μM for 4F2HC-LAT1 and 520 ± 52 μM for 4F2HC-LAT2. Vascular endothelial and smooth muscle cells were shown to express transcripts for 4F2HC and for both LAT1 and LAT2. Transport of l-CSNO into red blood cells, endothelial cells, and smooth muscle cells was inhibited by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and by large neutral amino acids demonstrating functional system L transporters in each cell type. Uptake of l-CSNO led to accumulation of cellular S-nitrosothiols and inhibition of both growth factor-induced ERK phosphorylation and TNF-α-mediated IκB degradation. Similar effects were seen when cells were incubated simultaneously with S-nitrosoalbumin and l-cysteine but not with d-cysteine or with S-nitrosoalbumin alone. In each case, nitrosylation of proteins and cellular responses were blocked by BCH. Together, these data suggest that transmembrane movement of nitric oxide (NO) equivalents from the plasma albumin NO reservoir is mediated by cysteine, which serves as a carrier. The mechanism requires transnitrosylation from S-nitrosoalbumin to free cysteine and activity of system L transporters, thereby providing a unique pathway for cellular responses to S-nitrosoalbumin.

Download Full-text

Heteromeric amino acid transporters: biochemistry, genetics, and physiology

AJP Renal Physiology ◽

10.1152/ajprenal.2001.281.6.f995 ◽

2001 ◽

Vol 281 (6) ◽

pp. F995-F1018 ◽

Cited By ~ 120

Author(s):

Josep Chillarón ◽

Ramón Roca ◽

Alfonso Valencia ◽

Antonio Zorzano ◽

Manuel Palacín

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Human Genetics ◽

Disulfide Bridge ◽

Transport Systems ◽

Cell Physiology ◽

Amino Acid Transporters ◽

Acid Transport ◽

Membrane Glycoproteins ◽

Cell Functions

The heteromeric amino acid transporters (HATs) are composed of two polypeptides: a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulfide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins. The HSHATs have been known since 1992, and the LSHATs have been described in the last three years. HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y+L isoforms, asc, x[Formula: see text], and b0,+). Members of the HAT family are the molecular bases of inherited primary aminoacidurias cystinuria and lysinuric protein intolerance. In addition to the role in amino acid transport, one HSHAT [the heavy subunit of the cell-surface antigen 4F2 (also named CD98)] is involved in other cell functions that might be related to integrin activation. This review covers the biochemistry, human genetics, and cell physiology of HATs, including the multifunctional character of CD98.

Download Full-text

The Arabidopsis paraquat resistant1 mutant accumulates leucine upon dark treatment

Botany ◽

10.1139/cjb-2016-0321 ◽

2017 ◽

Vol 95 (7) ◽

pp. 751-761 ◽

Cited By ~ 1

Author(s):

Rowshon A. Begam ◽

Allen G. Good

Keyword(s):

Amino Acid ◽

Growth Medium ◽

Homo Sapiens ◽

In Silico Analysis ◽

Amino Acid Transporters ◽

Wild Type ◽

Knockout Mutant ◽

Amino Acid Permease ◽

Polyamine Transport ◽

Uptake Studies

The Arabidopsis paraquat resistant1 (PAR1) was classified as L-type amino acid transporter 4 (LAT4) based on a phylogenetic analysis of selected genes from Saccharomyces cerevisiae, Arabidopsis thaliana (L.) Heynh, and Homo sapiens that clustered LAT4 with four other members as a LAT family in Arabidopsis. In silico analysis of the Arabidopsis LATs identified an amino acid permease domain and motifs that are common in amino acid transporters. However, their role in amino acid transport remained to be studied. A knockout mutant for PAR1/LAT4 gene, reported here as par1-5, showed significantly altered growth compared with wild type on leucine-containing growth medium. Mutant par1-5 seedlings showed reduced biomass compared with wild type on nitrate-containing Murashige and Skoog growth medium, which was further reduced when grown on medium containing nitrate and leucine. Radio-labelled leucine uptake studies using leaf protoplasts and seedlings showed increased accumulation of leucine in par1-5 mutants compared with wild type. Increased accumulation of leucine in par1-5 was detected when seedlings or protoplasts were treated in the dark prior to isotopic feeding. These studies suggest that the PAR1/LAT4 protein, in addition to its ability to mediate paraquat and polyamine transport, possess leucine transport activity that is regulated by physiological conditions such as dark induction.

Download Full-text

Replication kinetics of a CHIKV Asian strain with 76 aa duplication in the nsP3 HVD, in comparison to the wild-type Asian and ECSA strains

Access Microbiology ◽

10.1099/acmi.ac2020.po0797 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Nurshariza Abdullah ◽

Hong Wai Tham ◽

Nafees Abdullah ◽

Vinod RMT Balasubramaniam ◽

I-Ching Sam ◽

...

Keyword(s):

Amino Acid ◽

Preliminary Finding ◽

Wild Type ◽

Replication Rate ◽

Asian Strain ◽

Genotypic Analysis ◽

The Indian Ocean ◽

Kinetics Of ◽

Type Strains

Introduction: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus which causes fever, rash and polyarthralgia. CHIKV has expanded its circulating regions to the Indian Ocean islands, Europe, Americas and Southeast Asia. Two CHIKV lineages, the ASIAN and ECSA are circulating in Malaysia. In 2009, a CHIKV strain with a 76 amino acid (aa) duplication in its nsP3 hypervariable domain (HVD), identified as CHIKvASIAN09MUM-Dup+ was isolated from a patient co-infected with DENV-2. Indels and duplication have been found in many other alphaviruses, and suggested to play a role in the survivality of the viruses. Objectives: We aim to compare and relate the replication kinetics and virulency in-vitro of CHIKvASIAN09MUM-Dup+ with the wild-type Asian and ECSA strains. Methods & Results: Genotypic analysis was conducted on three CHIKV strains in Malaysia, the CHIKvASIAN06UM-Dup-, CHIKvECSA08UM-Dup- and CHIKvASIAN09MUM-Dup+. We found that CHIKvASIAN09MUM-Dup+ has significant low replication rates in Vero, C6/36 and Rhabdosarcoma cells as compared to the wild-type strains. The highest titers were reached by CHIKvASIAN09MUM-Dup+ in all cells are 6.5 to 6.75 log10 TCID50/mL, which is 100 fold lower compared to the wild-type strains. Conclusion: The significantly low replication rate of Dup+ strain in all the cells, maybe suggestive to be due to co-infection and co-existence with DENV, where the aa duplication may play a role in overcoming competitive suppression. This preliminary finding agrees with reported events, where alphaviruses use insertion, deletion and duplication of amino acid in nsP3 HVD as strategies to influence replication in host, viral virulency, pathogenesis and survivality for evolution adaptation.

Download Full-text

Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray, Dasyatis sabina

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00739.2005 ◽

2006 ◽

Vol 291 (3) ◽

pp. R844-R853 ◽

Cited By ~ 11

Author(s):

Michael G. Janech ◽

Wayne R. Fitzgibbon ◽

Mark W. Nowak ◽

Donald H. Miller ◽

Richard V. Paul ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Single Gene ◽

Functional Characterization ◽

Amino Acid Residues ◽

Urea Transporter ◽

Coding Region ◽

Acid Protein ◽

Atlantic Stingray ◽

Rapid Amplification

The cloning of cDNAs encoding facilitated urea transporters (UTs) from the kidneys of the elasmobranchs indicates that in these fish renal urea reabsorption occurs, at least in part, by passive processes. The previously described elasmobranch urea transporter clones from shark (shUT) and stingray (strUT-1) differ from each other primarily because of the COOH-terminus of the predicted strUT-1 translation product being extended by 51-amino acid residues compared with shUT. Previously, we noted multiple UT transcripts were present in stingray kidney. We hypothesized that a COOH terminally abbreviated UT isoform, homologous to shUT, would also be present in stingray kidney. Therefore, we used 5′/3′ rapid amplification of cDNA ends to identify a 3′UTR-variant (strUT-1a) of the cDNA that encodes (strUT-1), as well as three, 3′UTR-variant cDNAs (strUT-2a,b,c) that encode a second phloretin-sensitive, urea transporter (strUT-2). The 5′UTR and the first 1,132 nucleotides of the predicted coding region of the strUT-2 cDNAs are identical to the strUT-1 cDNAs. The remainder of the coding region contains only five novel nucleotides. The strUT-2 cDNAs putatively encode a 379-amino acid protein, the first 377 amino acids identical to strUT-1 plus 2 additional amino acids. We conclude that 1) a second UT isoform is expressed in the Atlantic stingray and that this isoform is similar in size to the UT previously cloned from the kidney of the dogfish shark, and 2) at least five transcripts encoding the 2 stingray UTs are derived from a single gene product through alternative splicing and polyadenylation.

Download Full-text

Functional characterization of the alanine-serine-cysteine exchanger of Carnobacterium sp AT7

The Journal of General Physiology ◽

10.1085/jgp.201812195 ◽

2019 ◽

Vol 151 (4) ◽

pp. 505-517 ◽

Cited By ~ 5

Author(s):

Paola Bartoccioni ◽

Joana Fort ◽

Antonio Zorzano ◽

Ekaitz Errasti-Murugarren ◽

Manuel Palacín

Keyword(s):

Amino Acid ◽

Molecular Mechanisms ◽

Structural Information ◽

Functional Characterization ◽

Physiological Role ◽

Cell Uptake ◽

Amino Acid Transporters ◽

Inhibitor Selectivity ◽

Substrate Binding Sites

Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites—a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.

Download Full-text