<i>Escherichia coli abg</i>
            Genes Enable Uptake and Cleavage of the Folate Catabolite
            <i>p</i>
            -Aminobenzoyl-Glutamate

Carter, Eric L.; Jager, L.P.; Gardner, Lars; Hall, Christel; Willis, Stacey; Green, Jacalyn M.

doi:10.1128/jb.01940-06

Cited by 36 publications

(55 citation statements)

References 28 publications

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“…The mutants expressed the aminopeptidases PepE and PepP. The aminobenzoylglutamate utilization protein AbgA is part of the abg operon involved in the catabolism of folate, a compound essential for the synthesis of DNA, RNA and amino acids (Carter et al, 2007). The deoxyribose-phosphate aldolase DeoC is known to be important for the catabolism of deoxyribonucleosides (Han et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

et al. 2009

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Proteus mirabilis causes urinary tract infections (UTIs) in individuals requiring long-term indwelling catheterization. The pathogenesis of this uropathogen is mediated by a number of virulence factors and the formation of crystalline biofilms. In addition, micro-organisms have evolved complex systems for the acquisition of nutrients, including the phosphate-specific transport system, which has been shown to be important in biofilm formation and pathogenesis. A functional Pst system is important during UTIs caused by P. mirabilis HI4320, since transposon mutants in the PstS periplasmic binding protein and the PstA permease protein were attenuated in the CBA mouse model of UTI. These mutants displayed a defect in biofilm formation when grown in human urine. This study focuses on a comparison of the proteomes during biofilm and planktonic growth in phosphate-rich medium and human urine, and microscopic investigations of biofilms formed by the pst mutants. Our data suggest that (i) the Dpst mutants, and particularly the DpstS mutant, are defective in biofilm formation, and (ii) the proteomes of these mutants differ significantly from that of the wild-type. Therefore, since the Pst system of P. mirabilis HI4320 negatively regulates biofilm formation, this system is important for the pathogenesis of these organisms during complicated UTIs. INTRODUCTIONProteus mirabilis -a member of the family Enterobacteriaceae -is the most common aetiological agent responsible for complicated urinary tract infections (UTIs) (Mobley, 1996). Subpopulations at higher risk for infection by this pathogen include those with long-term indwelling catheterization as well as those with structural and functional abnormalities within the urinary tract (Mobley, 1996). Clinical syndromes associated with P. mirabilis include cystitis and pyelonephritis, with possible complications from stone formation and bacteraemia (Mobley, 1996). These micro-organisms survive in the urinary tract by virtue of their production of a battery of virulence factors, including urease, flagella, fimbriae, haemolysin and IgA protease (Belas, 1996;Mobley et al., 1994;Musher et al., 1975;Peerbooms et al., 1984; Walker et al., 1999), and formation of crystalline biofilm on indwelling catheters (Stickler et al., 1993).Biofilm formation -one of the most important mechanisms of pathogenicity of this micro-organism in the urinary tract -may be defined as a community of surface-attached bacteria encased in an extracellular matrix consisting of secreted carbohydrates, proteins and DNA. Indeed, biofilms have been associated with the virulence of a number of pathogens (Castelli et al., 2006). Organisms in these communities display phenotypic differences from planktonic cells, including a slower rate of growth and increased resistance to antibiotics (Lewis, 2001;Stoodley et al., 2002). P. mirabilis biofilms in urine are unique in that the extracellular polysaccharide matrix is enmeshed with struvite and carbonate apatite crystals (Jones et al., 2006).Abbreviations: CLSM, confocal ...

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Section: Discussionmentioning

confidence: 99%

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

et al. 2009

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“…The E. coli abgT gene encodes a cryptic transporter of pABAglutamate (a folate breakdown product) that is activated by point mutations in its promoter region (25,41,42), making it a prime suspect to explain the interference problem. We therefore constructed a pabA abgT strain and compared its propensity to grow in liquid minimal medium plus 5-FHTF with that of the parent pabA strain (Fig.…”

Section: Construction and Validation Of Anmentioning

confidence: 99%

Identification of Transport-critical Residues in a Folate Transporter from the Folate-Biopterin Transporter (FBT) Family

Eudes

Kunji

Noiriel

et al. 2010

Journal of Biological Chemistry

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The Synechocystis Slr0642 protein and its plastidial Arabidopsis (Arabidopsis thaliana) ortholog At2g32040 belong to the folate-biopterin transporter (FBT) family within the major facilitator superfamily. Both proteins transport folates when expressed in Escherichia coli. Because the structural requirements for transport activity are not known for any FBT protein, we applied mutational analysis to identify residues that are critical to transport and interpreted the results using a comparative structural model based on E. coli lactose permease. Folate transport was assessed via the growth of an E. coli pabA abgT strain, which cannot synthesize or take up folates or p-aminobenzoylglutamate. In total, 47 residues were replaced with Cys or Ala. Mutations at 22 positions abolished folate uptake without affecting Slr0642 expression in membranes, whereas other mutations had no effect. Residues important for function mostly line the predicted central cavity and are concentrated in the core ␣-helices H1, H4, H7, and H10. The essential residue locations are consistent with a folate-binding site lying roughly equidistant from both faces of the transporter. Arabidopsis has eight FBT proteins besides At2g32040, often lacking conserved critical residues. When six of these proteins were expressed in E. coli or in Leishmania folate or pterin transporter mutants, none showed evidence of folate or pterin transport activity, and only At2g32040 was isolated by functional screening of Arabidopsis cDNA libraries in E. coli. Such negative data could reflect roles in transport of other substrates. These studies provide the first insights into the native structure and catalytic mechanism of FBT family carriers.

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“…COLI p-AMINOBENZOYL-GLUTAMATE HYDROLASE 2411 (4). AbgA and AbgB comprise subunits of a manganese-dependent enzyme that hydrolyzes PABA-GLU.…”

Section: Vol 192 2010mentioning

confidence: 99%

“…Previously, we had found that wild-type cells transformed with a high-copy-number plasmid carrying abgT demonstrated saturable uptake of PABA-GLU (K T [transport constant] ϭ 123 M); control cells harboring the vector alone demonstrated negligible uptake (4). Tritiated PABA-GLU taken in by cells expressing large amounts of AbgT was not rapidly metabolized to a form that was trapped in the cell, as addition of nonradioactive PABA-GLU to these cells resulted in rapid loss of intracellular label.…”

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Purification and Characterization of the Folate Catabolic Enzyme p -Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

et al. 2010

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The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (ϳ53-kDa and ϳ47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a K m value for PABA-GLU of 60 ؎ 0.08 M and a specific activity of 63,300 ؎ 600 nmol min ؊1 mg ؊1 . Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.Reduced derivatives of folic acid are required for biosynthesis of DNA, RNA, amino acids, and other important cellular components (14). Folic acid is an essential dietary supplement for humans, while both microorganisms and plants can synthesize this vitamin de novo. The Escherichia coli folic acid biosynthetic pathway is composed of proteins encoded by genes scattered across the chromosome (9); these genes appear to be constitutively expressed at low levels (26,27). The genes and enzymes involved in folate catabolism in E. coli remain largely unidentified.The abg region of E. coli was first identified in a search for mutants able to grow on folic acid in order to circumvent paminobenzoate (PABA) auxotrophy; characterization showed that the auxotrophs were utilizing the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU), not folic acid ( Fig. 1) (11). The original mutations were point mutations in the intergenic region between abgA and abgR, and it was hypothesized that this resulted in increased expression of the abgABT genes. The abg region, named for enhanced growth on p-aminobenzoyl-glutamate, was mapped to 30 min and was found to consist of a potential operon including abgA, abgB, and abgT. Divergently oriented from abgABT, abgR encodes AbgR, which has homology to LysR-type regulatory proteins (21). Sequence analysis of the putative gene products revealed that AbgA and AbgB were similar to one another and to aminoacyl aminohydrolases and that AbgT was similar to transport proteins (11).Previously, we had found that wild-typ...

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Escherichia coli abg Genes Enable Uptake and Cleavage of the Folate Catabolite p -Aminobenzoyl-Glutamate

Cited by 36 publications

References 28 publications

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation

Identification of Transport-critical Residues in a Folate Transporter from the Folate-Biopterin Transporter (FBT) Family

Purification and Characterization of the Folate Catabolic Enzyme p -Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

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