Chlamydia and chlamydial control of gene expression


Fig 33. This continuing presentation © Peter Timms and Sarah Mathews November 2002. Figure shows the typical way in which bacterial genes are arranged, with a promoter site to which the sigma factor - RNA polymerase complex binds, and a region encoding a ribosome binding site (RBS).	Fig 34. A comparison of the nucleotide sequences for various chlamydial genes of putative promoters recognised by the chlamydial sigma factor 66. The consensus promoter sequence recognised by the related E. coli sigma factor 70 is also shown.	Fig 35. Bioinformatics applied to the search for chlamydial promoters in the classic -35 10 region upstream of chlamydial genes in the C. trachomatis serovar D genome sequence. Known chlamydial sequences in this non protein coding region are compared with a matrix encompassing knowledge about the proportion of individual nucelotides in the E. coli -35 -10 promoter region. It is known that AT rich cis-acting DNA elements located immediately downstream of the -35 hexamer are required for high-level transcription of six chlamydial promoters. The AT rich sequence may extend the -35 promoter hexamer, act as a trans-activator binding site, or alter promoter DNA curvature to increase the initiation of transcription [Tan et al., 1998; Schaumberg & Tan 2000; Timms & Mathews 2002].	Fig 36. The matrix pattern searching methodology

Fig 37. An example of pattern searching for one particular promoter hexamer.	Fig 38. The methodology expanded.	Fig 39. Properties of the basic pattern searching algorithm.	Fig 40. Chlamydial genome projects already completed, from which data for bioinformatic identification of putative chlamydial promoters may be obtained. NB: Parachlamydia are also being sequenced.

Fig 41. The full bioinformatic pattern matrix methodology as it can be applied to multiple Chlamydiales genome sequences.	Fig 42. An example of the comparative alignment of non coding regions of chlamydial genes, encompassing the -35, -10 and +1 sites as well as the A/T spacer region which frequently augments chlamydial gene transcription.	Fig 43. Predictive value of the matrix bioinformatic approach. Once putative promoters have been identified, the chlamydial genome sequences may be searched for examples of other genes with those sequences.	Fig 44. The structure of chlamydial sigma factor 28, showing some of its distinguishing features.

Fig 45. Promoters and genes recognised by chlamydial sigma 28 include the heat shock responsive fliA.	Fig 46. The structure of chlamydial sigma factor 54	Fig 47. Promoters or genes recognised by sigma 54 in other bacteria.	Fig 48. Transcription initiation by sigma 54 is different to the basic eubacterial model for sigma 70 shown in Fig 25. Firstly sigma 54 binds to the promoter in the absence of core enzyme and recruits the core to form a stable closed complex. Open complex formation occurs when the RNA polymerase is activated by an enhancer binding protein which binds upstream of the promoter. In most cases this activation is mediated by curves in the DNA generated by integration host factor binding, IHB.

Fig 49. There are chlamydial homologues for all the genes required for sigma 54 induced transcription. These include RtoN, NtrC and IHB.	Fig 50. Identification of sigma 54 promoters in the chlamydial genome. Five genes had putative binding sites in their upstream sequences for IHB and for NtrC. See Mathews & Timms, 2000 and Wan et al., 2002.	Fig 51. Mapping the transcriptional start sites of four new candidate promoters. Using a fluorescent primer extension assay the 5' end of the transcripts, represented by the peaks, mapped to the promoters.	Fig 52. Having confirmed the sigma 54 promoters, equivalent sequences were sort in the genomic sequences of various chlamydiae. Interestingly the sigma 54 promoter was not conserved in the htrA or acpS sequences. This may be explained (Fig 53) by the arrangement of open reading frames.

Fig 53. The arrangement of open reading frames for genes with the conserved lpxA or nifS promoters in all genome sequences examined was the same. However for htrA and acpS the open reading frame arrangement was different, particularly for acpS.	Fig 54. Using the motif discovery tool MEME, the 350 base pairs upstream of the 6 s54 promoters identified were examined and two motifs were identified as shown. All sequences contained one of both of these motifs. The position of the predicted binding site for IHB relative to the promoters is shown.	Fig 55. Real time pcr was then used to identify the time of expression of all the genes with s54 promoters relative to the developmental cycle. Here the expression of rpoN is shown.	Fig 56. Expression of lpxA and nifS starts at about the same time as rpoN but is sustained at high level.

Fig 57. Expression of htrA and acpS	Fig 58. Other genes with s54 promoters were only expressed at low	Fig 59. Summary of genes controlled by sigma 54 in chlamydiae.	Fig 60. Summary of sigma 54 expression findings.

Fig 61.Summary of the comparative genomics of sigma 54 genes.	Fig 62. What is the overall importance of sigma factors in controlling gene expression?	Fig 63. There are between 850 - 1020 chlamydial genes and around 170 operons arranged in various regulons.	Fig 64. They may be as many as 500 individually regulated units.

Fig 65. It is unclear how many chlamydial genes may be regulated via multiple promoters.	Fig 66. Number of genes known to be regulated by each of the three chlamydial sigma factors versus year.	Fig 67. There may be various levels of transcriptional control operating on the same gene.	Fig 68. Diagram of the various levels of transcriptional control which might result, for example, from an environmental signal.

Fig 69. Table showing the rough relationship between bacterial genome size and the number of sigma factors and transcriptional complexity.	Fig 70. What transcriptional controls might be expected in chlamydiae?	Fig 71. Summary of the possible levels of gene regulation in chlamydiae.	Fig 72. Methods likely to bring greater insight into chlamydial gene regulation in the near future.

Fig 73. Acknowledgements to colleagues working with the QUT team.

Control of chlamydial gene expression

Presentation by Peter Timms and Sarah Mathews (QUT, Brisbane) Part 2.

References