The single extracytoplasmic-function sigma factor of Xylella fastidiosa is involved in the heat shock response and presents an unusual regulatory mechanism

Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA, predicted to encode an extracytoplasmic function (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa sigma(E) regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 sigma(E)-dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative sigma(E)-dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like other ECF sigma factors, rpoE and rseA were shown to comprise an operon in X. fastidiosa, together with a third open reading frame (XF2241). However, upon heat shock, rpoE expression was not induced, while rseA and XF2241 were highly induced at a newly identified sigma(E)-dependent promoter internal to the operon. Therefore, unlike many other ECF sigma factors, rpoE is not autoregulated but instead positively regulates the gene encoding its putative anti-sigma factor.     

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR

Rhodobacter sphaeroides sigma(E) is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R.sphaeroides, sigma(E) activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits sigma(E) activity. We found that formation of the sigma(E):ChrR complex inhibits the ability of sigma(E) to form a stable complex with core RNA polymerase. Since the sigma(E):ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in sigma(E) that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of sigma(E) that either increased or decreased the sensitivity of sigma(E) for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of sigma(E) to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the sigma(70) superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of sigma(E) interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.     

部分革兰氏阴性菌胞外功能 sigma因子结构与调控铁离子的利用机制

铁离子是大多数细菌生存所必需的一种营养物质,但摄入过多的铁离子也会对细菌造成损伤。因此,细菌对铁离子的摄取受到严格调控。革兰氏阴性菌对铁离子的摄取主要受Fur (ferric uptake regulator) 蛋白和σ（sigma）因子的调控。σ因子是RNA聚合酶的可解离亚基,能使RNA聚合酶结合到基因的启动子区域,从而引起基因转录。因此,σ因子在原核生物转录起始过程中必不可少。细菌中存在多种σ因子,参与铁离子调控的σ因子即是胞外功能σ因子（extra cytoplasmic function sigma factor, ECF sigma factor）。通常,胞外功能σ因子活性可被抗σ因子（anti sigma factor）抑制。当受到外界环境信号的刺激,σ因子与抗σ因子解离,从而使σ因子活化并结合RNA聚合酶核心酶形成全酶,引起目的基因的转录。本文将就胞外功能σ因子在σ因子家族中的分类地位、结构特点以及对3价铁离子和血红素的转运调控机制作一综述。     

Alternative sigma factors in the free state are equilibrium mixtures of open and compact conformations

Conformational switching upon core RNA polymerase binding is an integral part of functioning of bacterial sigma factors. Here, we have studied dynamical features of two alternative sigma factors. A study of fluorescence resonance energy transfer and hydrodynamic measurements in Escherichia coli σ(32) suggest a compact shape like those found in complex with anti-sigma factors. On the other hand, the fluorescence anisotropy of probes attached to different regions of the protein and previous hydrogen exchange measurements suggest significant internal flexibility, particularly in the C-terminal half and region 1. In a homologous sigma factor, σ(F) of Mycobacterium tuberculosis, emission spectra and fluorescence resonance energy transfer between the single tryptophan (W112) and probes placed in different regions suggest a compact conformation for a major part of the N-terminal half encompassing region 2 and the flexible C-terminal half. Fluorescence anisotropy measurements suggest significant flexibility in the C-terminal half and region 1, as well. Thus, free alternative sigma factors may be in equilibrium between two conformations: a compact one in which the promoter interacting motifs are trapped in the wrong conformation and another less abundant one with a more open and flexible conformation. Such flexibility may be important for promoter recognition and interaction with many partner proteins.     

Mutational analysis of an extracytoplasmic-function sigma factor to investigate its interactions with RNA polymerase and DNA

The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.