The Repressor Function of the Chlamydia Late Regulator EUO Is Enhanced by the Plasmid-Encoded Protein Pgp4.

A critical step in intracellular Chlamydia infection is the production of infectious progeny through the expression of late genes. This differentiation step involves conversion from a reticulate body (RB), which is the replicating form of the bacterium, into an elementary body (EB), which is the developmental form that spreads the infection to a new host cell. EUO is an important chlamydial transcription factor that controls the expression of late genes, but the mechanisms that regulate EUO are not known. We report that a plasmid-encoded protein, Pgp4, enhanced the repressor activity of EUO. Pgp4 did not function as a transcription factor because it did not bind or directly modulate transcription of its target promoters. Instead, Pgp4 increased the ability of EUO to bind and repress EUO-regulated promoters in vitro and physically interacted with EUO in pulldown assays with recombinant proteins. We detected earlier onset of EUO-dependent late gene expression by immunofluorescence microscopy in Pgp4-deficient C. trachomatis and C. muridarum strains. In addition, the absence of Pgp4 led to earlier onset of RB-to-EB conversion in C. muridarum These data support a role for Pgp4 as a negative regulator of chlamydial transcription that delays late gene expression. Our studies revealed that Pgp4 also has an EUO-independent function as a positive regulator of chlamydial transcription.IMPORTANCEChlamydia trachomatis is an important human pathogen that causes more than 150 million active cases of genital and eye infection in the world. This obligate intracellular bacterium produces infectious progeny within an infected human cell through the expression of late chlamydial genes. We showed that the ability of a key chlamydial transcription factor, EUO, to repress late genes was enhanced by a plasmid-encoded protein, Pgp4. In addition, studies with Chlamydia Pgp4-deficient strains provide evidence that Pgp4 delays late gene expression in infected cells. Thus, Pgp4 is a novel regulator of late gene expression in Chlamydia through its ability to enhance the repressor function of EUO.

Regulation of Chlamydia Gene Expression by Tandem Promoters with Different Temporal Patterns.

UNLABELLED: Chlamydia is a genus of pathogenic bacteria with an unusual intracellular developmental cycle marked by temporal waves of gene expression. The three main temporal groups of chlamydial genes are proposed to be controlled by separate mechanisms of transcriptional regulation. However, we have noted genes with discrepancies, such as the early gene dnaK and the midcycle genes bioY and pgk, which have promoters controlled by the late transcriptional regulators EUO and σ(28). To resolve this issue, we analyzed the promoters of these three genes in vitro and in Chlamydia trachomatis bacteria grown in cell culture. Transcripts from the σ(28)-dependent promoter of each gene were detected only at late times in the intracellular infection, bolstering the role of σ(28) RNA polymerase in late gene expression. In each case, however, expression prior to late times was due to a second promoter that was transcribed by σ(66) RNA polymerase, which is the major form of chlamydial polymerase. These results demonstrate that chlamydial genes can be transcribed from tandem promoters with different temporal profiles, leading to a composite expression pattern that differs from the expression profile of a single promoter. In addition, tandem promoters allow a gene to be regulated by multiple mechanisms of transcriptional regulation, such as DNA supercoiling or late regulation by EUO and σ(28). We discuss how tandem promoters broaden the repertoire of temporal gene expression patterns in the chlamydial developmental cycle and can be used to fine-tune the expression of specific genes. IMPORTANCE: Chlamydia is a pathogenic bacterium that is responsible for the majority of infectious disease cases reported to the CDC each year. It causes an intracellular infection that is characterized by coordinated expression of chlamydial genes in temporal waves. Chlamydial transcription has been shown to be regulated by DNA supercoiling, alternative forms of RNA polymerase, and transcription factors, but the number of transcription factors found in Chlamydia is far fewer than the number found in most bacteria. This report describes the use of tandem promoters that allow the temporal expression of a gene or operon to be controlled by more than one regulatory mechanism. This combinatorial strategy expands the range of expression patterns that are available to regulate chlamydial genes.

The transcriptional repressor EUO regulates both subsets of Chlamydia late genes.

The pathogenic bacterium Chlamydia replicates in a eukaryotic host cell via a developmental cycle marked by temporal waves of gene expression. We have previously shown that late genes transcribed by the major chlamydial RNA polymerase, σ(66) RNA polymerase, are regulated by a transcriptional repressor EUO. We now report that EUO also represses promoters for a second subset of late genes that are transcribed by an alternative polymerase called σ(28) RNA polymerase. EUO bound in the vicinity of six σ(28) -dependent promoters and inhibited transcription of each promoter. We used a mutational analysis to demonstrate that the EUO binding site functions as an operator that is necessary and sufficient for EUO-mediated repression of σ(28) -dependent transcription. We also verified specific binding of EUO to σ(66) -dependent and σ(28) -dependent promoters with a DNA immunoprecipitation assay. These findings support a model in which EUO represses expression of both σ(66) -dependent and σ(28) -dependent late genes. We thus propose that EUO is the master regulator of late gene expression in the chlamydial developmental cycle.

The early gene product EUO is a transcriptional repressor that selectively regulates promoters of Chlamydia late genes.

The obligate intracellular bacterium Chlamydia has an unusual developmental cycle in which there is conversion between two forms that are specialized for either intracellular replication or propagation of the infection to a new host cell. Expression of late chlamydial genes is upregulated during conversion from the replicating to the infectious form, but the mechanism for this temporal regulation is unknown. We found that EUO, which is expressed from an early gene, binds to two sites upstream of the late operon omcAB, but only the downstream site was necessary for transcriptional repression. Using gel shift and in vitro transcription assays we showed that EUO specifically bound and repressed promoters of Chlamydia trachomatis late genes, but not early or mid genes. These findings support a role for EUO as a temporal repressor that negatively regulates late chlamydial genes and prevents their premature expression. The basis of this specificity is the ability of EUO to selectively bind promoter regions of late genes, which would prevent their transcription by RNA polymerase. Thus, we propose that EUO is a master regulator that prevents the terminal differentiation of the replicating form of chlamydiae into the infectious form until sufficient rounds of replication have occurred.

Genetic analysis of the nitrogen assimilation control protein from Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae is a typical LysR-type transcriptional regulator (LTTR) in many ways. However, the lack of a physiologically relevant coeffector for NAC and the fact that NAC can carry out many of its functions as a dimer make NAC unusual among the LTTRs. In the absence of a crystal structure for NAC, we analyzed the effects of amino acid substitutions with a variety of phenotypes in an attempt to identify functionally important features of NAC. A substitution that changed the glutamine at amino acid 29 to alanine (Q29A) resulted in a NAC that was seriously defective in binding to DNA. The H26D substitution resulted in a NAC that could bind and repress transcription but not activate transcription. The I71A substitution resulted in a NAC polypeptide that remained monomeric. NAC tetramers can bind to both long and shorter binding sites (like other LTTRs). However, the absence of a coeffector to induce the conformational change needed for the switch from the former to the latter raised a question. Are there two conformations of NAC, analogous to the other LTTRs? The G217R substitution resulted in a NAC that could bind to the longer sites but had difficulty in binding to the shorter sites, and the I222R and A230R substitutions resulted in a NAC that could bind to the shorter sites but had difficulty in binding properly to the longer sites. Thus, there appear to be two conformations of NAC that can freely interconvert in the absence of a coeffector.

Developmental expression of dnaA is required for sporulation and timing of fruiting body formation in Myxococcus xanthus.

Inhibition of DNA replication within the first 6 h of development results in a block in the developmental programme in the social soil bacterium Myxococcus xanthus. We have interpreted these data to imply that M. xanthus requires a new round of DNA replication early in the developmental programme. To further understand the role of DNA replication during development in M. xanthus we focused on the regulation of dnaA which encodes the initiator protein of DNA replication. In this work, we demonstrate that immediately upon nutrient deprivation dnaA (MXAN1001) transcript levels decrease to 10-15% of vegetative levels and then transiently increase between 4 and 6 h post initiation. This expression is dependent on several early developmental regulators, including relA (MXAN3204), sigD (MXAN2957) and sdeK (MXAN1014). It is also dependent upon an 85 bp region located just upstream to the dnaA promoter. Our data suggest that while developmental dnaA expression is not essential for development, its expression allows for the proper timing and maximum efficiency of the sporulation process. In addition, we speculate that developmental control of dnaA expression may provide a mechanism for predetermination of cell fate during the differentiation process.

The LysR-type nitrogen assimilation control protein forms complexes with both long and short DNA binding sites in the absence of coeffectors.

Most LysR-type transcriptional regulators (LTTRs) function as tetramers when regulating gene expression. The nitrogen assimilation control protein (NAC) generally functions as a dimer when binding to DNA and activating transcription. However, at some sites, NAC binds as a tetramer. Like many LTTRs, NAC tetramers can recognize sites with long footprints (74 bp for the site at nac) with a substantial DNA bend or short footprints (56 bp for the site at cod) with less DNA bending. However, unlike other LTTRs, NAC can recognize both types of sites in the absence of physiologically relevant coeffectors, suggesting that the two conformers of the NAC tetramer (extended and compact) are interchangeable without the need for any modification to induce or stabilize the change. In order for NAC to bind as a tetramer, three interactions must exist: an interaction between the two NAC dimers and an interaction between each NAC dimer and its corresponding binding site. The interaction between one dimer and its DNA site can be weak (recognizing a half-site rather than a full dimer-binding site), but the other two interactions must be strong. Since the conformation of the NAC tetramer (extended or compact) is determined by the nature of the DNA site without the intervention of a small molecule, we argue that the coeffector that determines the conformation of the NAC tetramer is the DNA site to which it binds.

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication.

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB(A116V), was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37 degrees C. We demonstrated that at the nonpermissive temperature the DnaB(A116V) mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB(A116V) mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

Importance of tetramer formation by the nitrogen assimilation control protein for strong repression of glutamate dehydrogenase formation in Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae is a very versatile regulatory protein. NAC activates transcription of operons such as hut (histidine utilization) and ure (urea utilization), whose products generate ammonia. NAC also represses the transcription of genes such as gdhA, whose products use ammonia. NAC exerts a weak repression at gdhA by competing with the binding of a lysine-sensitive activator. NAC also strongly represses transcription of gdhA (about 20-fold) by binding to two separated sites, suggesting a model involving DNA looping. We have identified negative control mutants that are unable to exert this strong repression of gdhA expression but still activate hut and ure expression normally. Some of these negative control mutants (e.g., NAC(86ter) and NAC(132ter)) delete the C-terminal domain, thought to be required for tetramerization. Other negative control mutants (e.g., NAC(L111K) and NAC(L125R)) alter single amino acids involved in tetramerization. In this work we used gel filtration to show that NAC(86ter) and NAC(L111K) are dimers in solution, even at high concentration (NAC(WT) is a tetramer). Moreover, using a combination of DNase I footprints and gel mobility shifts assays, we showed that when NAC(WT) binds to two adjacent sites on a DNA fragment, NAC(WT) binds as a tetramer that bends the DNA fragment significantly. NAC(L111K) binds to such a fragment as two independent dimers without inducing the strong bend. Thus, NAC(L111K) is a dimer in solution or when bound to DNA. NAC(L111K) (typical of the negative control mutants) is wild type for every other property tested: (i) it activates transcription at hut and ure; (ii) it competes with the lysine-sensitive activator for binding at gdhA; (iii) it binds to the same sites at the hut, ure, nac, and gdhA promoters as NAC(WT); (iv) the relative affinity of NAC(L111K) for these sites follows the same order as NAC(WT) (ure > gdhA > nac > hut); (v) it induces the same slight bend as dimers of NAC(WT); and (vi) its DNase I footprints at these sites are indistinguishable from those of NAC(WT) (except for features ascribed to tetramer formation). The only two phenotypes we know for negative control mutants of NAC are their inability to tetramerize and their inability to cause the strong repression of gdhA. Thus, we propose that in order for NAC(WT) to exert the strong repression, it must form a tetramer that bridges the two sites at gdhA (similar to other DNA looping models) and that the negative control mutants of NAC, which fail to tetramerize, cannot form this loop and thus fail to exert the strong repression at gdhA.

Nitrogen regulation of the codBA (cytosine deaminase) operon from Escherichia coli by the nitrogen assimilation control protein, NAC.

Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. Beta-galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position -59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions -120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions -83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

Isolation of a negative control mutant of the nitrogen assimilation control protein, NAC, in Klebsiella aerogenes.

A negative control mutant of the nitrogen assimilation control protein, NAC, has been isolated. Mutants with the leucine at position 111 changed to a nonhydrophobic residue activate transcription from hut and ure promoters, but fail to repress gdhA expression. This failure does not result from failure to bind to either of the two sites required for gdhA repression, but the binding at those sites is altered in the mutant. It appears that the NAC negative control mutants fail to form the complex structures (probably tetramers) formed by wild-type NAC at the gdhA promoter.