Identification of a second <i>Mycobacterium tuberculosis</i> gene cluster encoding proteins of an ABC phosphate transporter

Braibant, Martine; Lefèvre, Philippe; Wit, L. De; Ooms, Josette; Peirs, Priska; Huygen, Kris; Wattiez, Ruddy

doi:10.1016/0014-5793(96)00953-2

Cited by 45 publications

(46 citation statements)

References 44 publications

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“…We have found that pstS-1 (Fig. 1) and pstS-2 (not shown) homologs exist in the genome of M. leprae with a similar organization to that described here for M. tuberculosis, whereas a pstS-3 M. leprae homolog is located at a distant site, within a permease gene cluster similar to the one found in M. tuberculosis (9). In the M. leprae genome, an additional fourth pstS-like gene was found, which we designated pstS-4.…”

Section: Discussionsupporting

confidence: 69%

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Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG

Lefèvre¹,

Braibant²,

Wit³

et al. 1997

J Bacteriol

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A gene encoding a protein homologous to the periplasmic ABC phosphate binding receptor PstS from Escherichia coli was cloned and sequenced from a gt11 library of Mycobacterium tuberculosis by screening with monoclonal antibody 2A1-2. Its degree of similarity to the E. coli PstS is comparable to those of the previously described M. tuberculosis phosphate binding protein pab (Ag78, Ag5, or 38-kDa protein) and another M. tuberculosis protein which we identified recently. We suggest that the three M. tuberculosis proteins share a similar function and could be named PstS-1, PstS-2, and PstS-3, respectively. Molecular modeling of their three-dimensional structures using the structure of the E. coli PstS as a template and their inducibility by phosphate starvation support this view. Recombinant PstS-2 and PstS-3 were produced and purified by affinity chromatography. With PstS-1, these proteins were used to demonstrate the specificity of three groups of monoclonal antibodies. Using these antibodies in flow cytometry and immunoblotting analyses, we demonstrate that the three genes are expressed and their protein products are present and accessible at the mycobacterial surface as well as in its culture filtrate. Together with the M. tuberculosis genes encoding homologs of the PstA, PstB, and PstC components we cloned before, the present data suggest that at least one, and possibly several, related and functional ABC phosphate transporters exist in mycobacteria. It is hypothesized that the mycobacterial gene duplications presented here may be a subtle adaptation of intracellular pathogens to phosphate starvation in their alternating growth environments.

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Section: Discussionsupporting

confidence: 69%

“…We have recently cloned and sequenced a few other M. tuberculosis genes corresponding to two PstA, two PstC, and one PstB components of putative phosphate permeases (8)(9)(10). The three pstS genes are positioned in different orientations and are probably clustered in three potential adjacent operons controlled by different promoters (Fig.…”

Section: Discussionmentioning

confidence: 99%

Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG

Lefèvre¹,

Braibant²,

Wit³

et al. 1997

J Bacteriol

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“…Target DNA carrying mutations in the PhnF binding sites was created as described previously (19). Binding reactions were carried out in binding buffer (20 …”

Section: Methodsmentioning

confidence: 99%

“…While M. tuberculosis possesses multiple copies of genes encoding Pst systems (20,21), M. smegmatis contains only a single copy of the pstSCAB genes (4,22). It therefore appears that the PhnDCE transporter, regulated primarily by PhnF, offers M. smegmatis a mechanism that is an alternative to that of multiple Pst systems for the efficient uptake of P i under growth-limiting conditions.…”

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confidence: 99%

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

et al. 2014

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dBacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains.

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“…2F8-3 (anti-PstS-3 ; Braibant et al, 1996) Alkaline phosphatase conjugated anti-mouse or anti-rabbit immunoglobulins (Promega) were used at a dilution of 1\7500 and 1\5000 respectively. Purification of Hsp60-2b.…”

Section: Construction Of Prr3hsp60-2mentioning

confidence: 99%

Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG Hsp60 proteins, and to the Escherichia coli homologue GroEL

et al. 2000

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Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C 16 :0 , C 18 :0 and C 18 :1 as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins.

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Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter

Cited by 45 publications

References 44 publications

Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG

Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG Hsp60 proteins, and to the Escherichia coli homologue GroEL

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