A developmentally regulated chlamydial gene with apparent homology to eukaryotic histone H 1 ( Chlamydia trachomatis )

We have developed a method for the isolation of genes whose expression is developmentally regulated from the murine strain of Chlamydia trachomatis. Here we describe the identification of two developmental stage-specific genes, one of which is predicted to encode a 26-kDa lysineand alanine-rich protein that appears to be homologous to several eukaryotic histone H1 proteins. A substantial proportion ofthis homology relates to its distinctive amino acid composition. No sequence homology was observed between this protein and other bacterial "histone-like" chromosomal proteins, but homology does exist with two other recently described prokaryotic proteins. The protein is expressed late in chiamydial development, during the transition from reticulate bodies to elementary bodies. The basic nature of the protein predicts that it could bind DNA, and Southwestern blotting experiments confirm this finding. These properties are consistent with a role either in the regulation of late gene expression or in the compaction of the chlamydial genome. Chlamydia trachomatis is an obligate bacterial pathogen that displays an interesting developmental cycle involving two morphologically distinct forms. The spore-like, metabolically inactive elementary body (EB) is the extracellular infectious form. EBs attach to and enter eukaryotic cells; once within a host-derived cytoplasmic vacuole, they differentiate into metabolically active reticulate bodies (RBs). After replicating within this vacuole, the RBs redifferentiate into EBs, thus completing the developmental cycle (1). This life cycle has been well-described morphologically, and distinctive ultrastructural changes have been observed during the transition between the two developmental forms (1). In addition to significant differences in size and membrane permeability between EBs and RBs, chromatin organization varies markedly between the two forms. Notably, the DNA genome of RBs resembles that of other bacteria, with diffuse fine fibrils extending throughout the cell, whereas mature EBs have a discrete, condensed electrondense nucleoid that appears to be unique among prokaryotes (2). These changes and others that characterize the chlamydial developmental cycle are temporally regulated. We are interested in identifying and characterizing genes that are involved in these developmental transitions. As a first step toward this goal, we have developed a straightforward though laborious method for the identification of chlamydial genes whose expression is regulated either temporally or by environmental stimuli. We report here the cloning of two genes that are expressed specifically late in chlamydial development, during the transition from RBs to EBs. Surprisingly, characterization ofone of these genes reveals it to encode a small, basic protein that shows apparent homology to eukaryotic histone H1 proteins.§ A preliminary account of this work was presented at the 1990 International Chlamydia Symposium (Harrison Hot Springs, Vancouver, Canada) and is summarized in its proceedings (3). MATERIALS AND METHODS Nucleic Acid Preparation and Analysis. Chlamydial DNA from the mouse pneumonitis strain of C. trachomatis (MoPn) or the lymphogranuloma venereum strain (serovar L2) of C. trachomatis was prepared as described (4). Total RNA was prepared from infected cells (at the indicated times following MoPn infection) as described (5). Preparation and Screening of Chlamydial DNA Libraries. For preparation of a plasmid-based library, chlamydial DNA was digested with EcoRI and cloned into a pGEM7Zf vector (Promega) previously cleaved with EcoRI and dephosphorylated at the 5' termini. Individual insert-bearing clones were selected randomly and DNA was prepared from them by the minilysate method (6). Probes were made by nick-translation of individual miniprep DNAs and then used to probe Northern blots containing chlamydial RNA extracted from cells at various times postinfection or following ampicillin treatment or exposure to a brief heat shock (5). To minimize detection of contaminating host cell DNAs (4) in the screening process, DNA was prepared from chlamydia grown in the human cell line HeLa, while RNA was isolated from infected mouse L cells. The A-based library was prepared in AgtWES as described (4). DNA Sequencing. The dideoxy chain-termination method ofDNA sequencing (7) was carried out on doubleor singlestrandedDNA prepared from fragment-containing plasmid or phagemid vectors, pGEM7Zf, or pBluescript KS(+) or SK(+) (Stratagene). Sequencing reactions were primed with oligonucleotides (Promega) complementary to the SP6 and T7 promoter sequences of pGEMZf that flank the inserted DNA. Reactions were carried out using the Sequenase kit (IBI) following the manufacturer's instructions for sequencing using the dideoxy G and the dideoxy I reagents provided. Immunoblotting. SDS/polyacrylamide gels and Western blots were carried out as described (5). The anti-Hcl antiserum, a rabbit polyclonal antiserum raised to the purified 18-kDa protein from the L2 serovar of C. trachomatis (8), and the anti-KARP antibody [where KARP indicates lysine (K)and alanine (A)-rich protein], a mouse monoclonal antibody raised to purified 32-kDa protein from the L2 serovar of C. trachomatis (generously provided by Ted Hackstadt, Rocky Abbreviations: NBRF, National Biomedical Research Foundation; EB, elementary body; RB, reticulate body; hpi, hours postinfection; ORF, open reading frame; MoPn, mouse pneumonitis strain of C. trachomatis; KARP, lysine (K)and alanine (A)-rich protein. *Present address: Department of Biology, San Francisco State University, San Francisco, CA 94132. tTo whom reprint requests should be addressed. §The DNA sequences of the proteins discussed in this paper have been deposited in the GenBank data base (accession no. M86605). 2125 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 89 (1992) Mountain Laboratories, Hamilton, MT), were used at a 1:200 dilution. The immunoblots were probed with a second antibody [goat anti-mouse or goat anti-rabbit IgG conjugated to alkaline phosphatase, respectively (Promega)]. The Southwestern blots were carried out according to Wagar and Stephens (9) except that 105 cpm of probe made by nicktranslating either total MoPn DNA or Escherichia coli DNA was incubated with the Western blots at room temperature for 1-2 hr. A iC\ >\