Purification and N-terminal amino acid sequences of Chlamydia trachomatis histone analogs

Hackstadt, Ted

doi:10.1128/jb.173.21.7046-7049.1991

Cited by 20 publications

(16 citation statements)

References 18 publications

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“…C. trachomatis nucleoids were isolated from EBs by extraction with 2% Zwittergent 3-14 (Calbiochem) for 1 h at room temperature with rotation. This procedure yielded structures consisting of the nucleoid surrounded by the freely permeable disulfide-linked outer membrane complex (19). Nucleoid complexes were pelleted at 15,000 ϫ g for 5 min and washed at least three times with PBSE (50 mM sodium phosphate, pH 7.5͞150 mM NaCl͞1.5 mM EDTA) Deproteinated E. coli Extract.…”

Section: Methodsmentioning

confidence: 99%

Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis

Grieshaber

Fischer

Mead

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The chlamydial developmental cycle is characterized by an intracellular replicative form, termed the reticulate body, and an extracellular form called the elementary body. Elementary bodies are characterized by a condensed chromatin, which is maintained by a histone H1-like protein, Hc1. Differentiation of elementary bodies to reticulate bodies is accompanied by dispersal of the chromatin as chlamydiae become transcriptionally active, although the mechanisms of Hc1 release from DNA have remained unknown. Dissociation of the nucleoid requires chlamydial transcription and translation with negligible loss of Hc1. A genetic screen was therefore designed to identify chlamydial genes rescuing Escherichia coli from the lethal effects of Hc1 overexpression. CT804, a gene homologous to ispE, which encodes an intermediate enzyme of the non-mevalonate methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, was selected. E. coli coexpressing CT804 and Hc1 grew normally, although they expressed Hc1 to a level equivalent to that which condensed the chromatin of parent Hc1-expressing controls. Inhibition of the MEP pathway with fosmidomycin abolished IspE rescue of Hc1-expressing E. coli. Deproteinated extract from IspE-expressing bacteria caused dispersal of purified chlamydial nucleoids, suggesting that chlamydial histone-DNA interactions are disrupted by a small metabolite within the MEP pathway rather than by direct action of IspE. By partial reconstruction of the MEP pathway, we determined that 2-C-methylerythritol 2,4-cyclodiphosphate dissociated Hc1 from chlamydial chromatin. These results suggest that chlamydial histone-DNA interactions are disrupted upon germination by a small metabolite in the MEP pathway of isoprenoid biosynthesis.C hlamydia trachomatis is the leading cause of preventable blindness worldwide and a major cause of sexually transmitted disease in developed countries (1). Chlamydiae are bacterial obligate intracellular pathogens with a biphasic developmental cycle that alternates between infectious extracellular forms, termed elementary bodies (EBs), and intracellular replicative forms known as reticulate bodies (RBs) (2). The metabolically inert EBs are characterized by a condensed nucleoid structure. Within a few hours after infection the chromatin becomes dispersed as transcription is initiated and differentiation to the larger, metabolically active RBs begins (3). RBs replicate by binary fission until 18 to 48 h after infection, at which time they begin to differentiate back to EBs, a process typified by recompaction of the nucleoid.The DNA of EBs is held in a condensed state by the action of two histone H1 homologs, Hc1 and Hc2 (4-8). Hc1 is conserved among all chlamydiae, whereas Hc2 displays a variable molecular weight depending on the C. trachomatis serovar and is absent from some chlamydial species and strains (5). Both hctA and hctB, encoding Hc1 and Hc2, respectively, are transcribed late in the developmental cycle concomitant with RB differentiation back to EBs and nucleoid cond...

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Section: Methodsmentioning

confidence: 99%

Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis

Grieshaber

Fischer

Mead

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…RESULTS Molecular cloning of hctB. The marked similarity of the N-terminal amino acid sequences of purified Hc2 proteins of serotypes L2, D, and B (15) and that deduced for the 26-kDa KARP of C trachomatis MoPn (27) strongly suggested that these proteins were homologous. Recombinant Xgtll bacteriophage encoding Hc2 of C. trachomatis L2 were identified by high-stringency in situ plaque hybridization using a cloned DNA probe internal to the coding sequences for the KARP of strain MoPn.…”

mentioning

confidence: 98%

“…Immunoblot analysis of purified preparations of L2 RBs and EBs by using an Hc2-specific monoclonal antibody further confirmed that Hc2 is undetectable at the RB stage of chlamydial development (5). This family of proteins includes the lysine-and alanine-rich protein (KARP) of strain MoPn (27) as well as other proteins described previously (14)(15)(16), which in. serotype L2 have an apparent molecular mass of 32 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gels.…”

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confidence: 99%

Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity

1993

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Two DNA-binding proteins with similarity to eukaryotic histone H1 Chlamydia trachomatis is an obligate intracellular parasitic bacterium with a biphasic developmental cycle alternating between the extracellular, metabolically inactive elementary body (EB) form and the intracellular metabolically active reticulate body (RB) form that replicates within the eukaryotic host cell (24). The chlamydial cycle of infection begins with the uptake of an EB by a susceptible host cell into a membrane-bound vacuole. Within approximately 8 h, differentiation to the larger RB form begins, marked by the reduction of the disulfide-linked EB outer membrane complex and dispersal of the highly condensed EB nucleoid. RBs multiply by binary fission within the cellular inclusion until 24 to 36 h after their internalization, at which time the developmental transition from RB back to EB begins. Characteristic compaction of the nucleoid occurs, accompanied by oxidative disulfide cross-linking of the outer membrane complex to form the mechanically rigid, impermeable EB cell wall.

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“…Shown here is a silver-stained SDS-PAGE profile of fractions of a whole-cell lysate of C. burnetii (9mi/II), an octylglucopyranoside extract (OGP-Sup), and fractions eluted with increasing NaCl concentrations. C. burnetii was extracted with a mixture containing 100 mM sodium carbonate, 10 mM EDTA, and 1% octylglucopyranoside (pH 10.5), and Hq1 was purified by heparin-agarose affinity chromatography essentially as described previously (6). Hq1 bound heparin with high affinity and was eluted at a NaCl concentration of 1 M. The N-terminal amino acid sequence of the purified Hq1 was determined with an Applied Biosystems model 470A protein sequencer.…”

Section: Figmentioning

confidence: 99%

“…The affinity of the presumed histone-like proteins of C. burnetii for DNA suggested that they may be amenable to purification schemes developed for isolation of chlamydial histones. C. burnetii was extracted with a mixture of 100 mM sodium carbonate, 10 mM EDTA, and 1% octylglucopyranoside (pH 10.5), and Hq1 was purified by heparin-agarose affinity chromatography essentially as described previously (6). Hq1 bound heparin with high affinity and was eluted at a NaCl concentration of 1 M (Fig.…”

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confidence: 99%

A developmental stage-specific histone H1 homolog of Coxiella burnetii

Heinzen

Hackstadt

1996

J Bacteriol

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Two DNA-binding proteins have been detected in Coxiella burnetii by southwestern (DNA-protein) blotting. One of these, termed Hq1, is enriched in the small cell variant stage of the developmental cycle and displays compositional and primary amino acid sequence similarities to eukaryotic histone H1. C. burnetii appears to be another example of an intracellular parasite with morphologically distinct developmental forms whose nucleoid structure may be controlled by histone H1 homologs.

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Purification and N-terminal amino acid sequences of Chlamydia trachomatis histone analogs

Cited by 20 publications

References 18 publications

Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis

Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis

Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity

A developmental stage-specific histone H1 homolog of Coxiella burnetii

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