Processing of cell‐surface signalling anti‐sigma factors prior to signal recognition is a conserved autoproteolytic mechanism that produces two functional domains

Cell‐surface signalling (CSS) enables Gram‐negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti‐sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic function (ECF) sigma factor. Recent studies have demonstrated that RseP‐mediated proteolysis of the anti‐sigma factors is key to σ^ECF activation. Using the Pseudomonas aeruginosa FoxR anti‐sigma factor, we show here that RseP is responsible for the generation of an N‐terminal tail that likely contains pro‐sigma activity. Furthermore, it has been reported previously that this anti‐sigma factor is processed in two separate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the CSS signal. However, our results also indicate that this processing event is not essential for activity. In fact, we have identified functional CSS anti‐sigma factors that are not cleaved prior to signal perception. Together, our results indicate that CSS regulation can occur through both complete and initially processed anti‐sigma factors.     

Characterization of five novel Pseudomonas aeruginosa cell-surface signalling systems

Cell-surface signalling is a sophisticated regulatory mechanism used by Gram-negative bacteria to sense signals from outside the cell and transmit them into the cytoplasm. This regulatory system consists of an outer membrane-localized TonB-dependent receptor (TonB-dependent transducer), a cytoplasmic membrane-localized antisigma factor and an extracytoplasmic function (ECF) sigma factor. Pseudomonas aeruginosa contains 13 potential surface signalling systems of which only six have been studied in detail. In this work we have identified the regulons of five novel P. aeruginosa signalling systems. For that, the ECF sigmas PA0149, PA1912, PA2050, PA2093 and PA4896 have been overexpressed and their target gene candidates have been identified using DNA microarray, proteomic analysis, and/or lacZ reporter construct. All five ECF sigma factors control the production of one TonB-dependent transducer. Interestingly, two sigma factors, PA2050 and PA2093, regulate the synthesis of a second transducer. Furthermore, we show that although all these sigma factors seem to control putative (metal) transport systems, one of them also regulates the expression of P. aeruginosa pyocins. Finally, we also show that the PA1912-PA1911-PA1910 (designated FemI-FemR-FemA in this work) signalling system responds to the presence of the Mycobacterium siderophores mycobactin and carboxymycobactin and is involved in the utilization of these heterologous siderophores.     

Differential proteolysis of sigma regulators controls cell-surface signalling in Pseudomonas aeruginosa

Cell-surface signalling systems are widespread in Gram-negative bacteria. In these systems gene expression occurs following binding of a ligand, commonly a siderophore, to a receptor protein in the outer membrane. The receptor interacts with a sigma regulator protein that extends from the periplasm into the cytoplasm to control the activity of a cognate sigma factor. The mechanisms of signal transduction in cell-surface signalling systems have not been determined. Here we investigate signal transduction in the pyoverdine, ferrichrome and desferrioxamine siderophore systems of Pseudomonas aeruginosa. When pyoverdine is present the sigma regulator FpvR undergoes complete proteolysis resulting in activation of two sigma factors PvdS and FpvI and expression of genes for pyoverdine synthesis and uptake. When pyoverdine is absent subfragments of FpvR inhibit PvdS and FpvI. Similarly, subfragments of the sigma regulators FoxR and FiuR are formed in the absence of desferrioxamine and ferrichrome. These are much less abundant when the siderophores are present and downstream gene expression takes place. In all three systems RseP (MucP/YaeL) is required for complete proteolysis of the sigma regulator and sigma factor activity. These findings indicate that regulated proteolysis is a general mechanism for signal transduction in cell-surface signalling.     

Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis

Under iron-limiting conditions, Pseudomonas aeruginosa produces a siderophore called pyoverdine. Pyoverdine is secreted into the extracellular environment where it chelates iron, and the resulting ferri-pyoverdine complexes are transported back into the bacteria by a cell surface receptor protein FpvA. Pyoverdine also acts as a signalling molecule inducing the production of three secreted virulence factors. Binding of ferri-pyoverdine to FpvA transduces a signal to the periplasmic part of the membrane-spanning antisigma factor FpvR. The signal is transmitted to the cytoplasmic part of FpvR, which controls the activity of an extracytoplasmic family (ECF) sigma factor protein PvdS. This results in the production of the virulence factors pyoverdine, exotoxin A and PrpL endoprotease. Here, we show that a second divergent branch of this signalling pathway regulates the production of the FpvA protein. FpvR negatively regulates the activity of a second ECF sigma factor, FpvI, which is required for the synthesis of FpvA, and the presence of ferri-pyoverdine greatly increases the activity of FpvI so that production of FpvA is induced. To the best of our knowledge, this is the first example of a branched signalling system of this sort and the first example of an antisigma factor protein (FpvR) that directly regulates the activities of two different ECF sigma factor proteins (PvdS and FpvI).     

Recent insights into iron import by bacteria

Bacteria are confronted with a low availability of iron owing to its insolubility in the Fe3+ form or its being bound to host proteins. The bacteria cope with the iron deficiency by using host heme or siderophores synthesized by themselves or other microbes. In contrast to most other nutrients, iron compounds are tightly bound to proteins at the cell surfaces, from which they are further translocated by highly specific proteins across the cell wall of gram-positive bacteria and the outer membrane of gram-negative bacteria. Once heme and iron siderophores arrive at the cytoplasmic membrane, they are taken up across the cytoplasmic membrane by ABC transporters. Here we present an outline of bacterial heme and iron siderophore transport exemplified by a few selected cases in which recent progress in the understanding of the transport mechanisms has been achieved.     

Rapid evolution of a bacterial iron acquisition system

Under iron limitation, bacteria scavenge ferric (Fe³⁺) iron bound to siderophores or other chelates from the environment to fulfill their nutritional requirement. In gram‐negative bacteria, the siderophore uptake system prototype consists of an outer membrane transporter, a periplasmic binding protein and a cytoplasmic membrane transporter, each specific for a single ferric siderophore or siderophore family. Here, we show that spontaneous single gain‐of‐function missense mutations in outer membrane transporter genes of Bradyrhizobium japonicum were sufficient to confer on cells the ability to use synthetic or natural iron siderophores, suggesting that selectivity is limited primarily to the outer membrane and can be readily modified. Moreover, growth on natural or synthetic chelators required the cytoplasmic membrane ferrous (Fe²⁺) iron transporter FeoB, suggesting that iron is both dissociated from the chelate and reduced to the ferrous form within the periplasm prior to cytoplasmic entry. The data suggest rapid adaptation to environmental iron by facile mutation of selective outer membrane transporter genes and by non‐selective uptake components that do not require mutation to accommodate new iron sources.     

A phylogenomic study of the general stress response sigma factor sigmaB of Bacillus subtilis and its regulatory proteins

Regulation of expression of the general stress regulon of Bacillus subtilis is mediated by the activation of the alternative sigma factor sigmaB. Activation of sigmaB is accomplished by a complex regulatory network involving protein-protein interactions and reversible protein phosphorylation. PSI-BLAST searches were performed and phylogenetic trees for sigmaB and its regulatory proteins were constructed. Occurrence of sigmaB is restricted to a small group of gram-positive bacteria (Bacillus, Staphylococcus, Listeria). Related sigma factors also involved in stress responses are present in Mycobacterium tuberculosis, Streptomyces species and even in cyanobacteria (Synechocystis species). Putative regulatory proteins found in several other bacterial species can be broadly catagorized into three categories: Anti sigma factors, anti-anti sigma factors and phosphatases. Anti sigma factors are able to bind to sigma factors and are also kinases of anti sigma factor antagonists. Only in their nonphosphorylated state, these antagonists are able to bind to the anti sigma factor. Phosphorylated antagonists can be dephosphorylated by PP2C phosphatases. These phosphatases are of pivotal importance for activation of the sigma factor. Different phosphatases identified in this search contain a wide variety of domains found in signal transducing proteins (PAS/PAC, GAF, REC, HATase_c, HAMP). The HATPase_c domain found in several phosphatases most probably constitutes a serine/threonine kinase domain of anti sigma factors. Such proteins are most probably bifunctional anti-anti sigma factor kinases and phosphatases. The regulatory network of anti-anti sigma factors anti sigma factors and phosphatases is probably ancient and most likely evolved from a structurally similar network found in the Deinococcus radiodurans genome. In completely sequenced genomes of several bacterial species, some elements of the network are missing. The N-terminus of RsbU, a phosphatase activated in response to environmental stress exhibits similarities to a region in the beta chain of phenylalanyl-tRNA synthetases.     

An extracytoplasmic function sigma factor-mediated cell surface signaling system in Pseudomonas syringae pv. tomato DC3000 regulates gene expression in response to heterologous siderophores

The diversity of regulatory systems encoded by bacteria provides an indication of the variety of stresses and interactions that these organisms encounter in nature. We have been investigating how the plant pathogen Pseudomonas syringae pv. tomato DC3000 responds to iron limitation and have focused on the iron starvation (IS) sigma factors to identify regulon members and to explore the mechanistic details of genetic control for this class of regulators. In the study described in this report, we used chromatin immunoprecipitation paired with high-throughput sequencing (ChIP-Seq) to screen the genome for locations associated with binding of the P. syringae IS sigma factor PSPTO_1203. We used multiple methods to demonstrate differential regulation of two genes identified in the ChIP-Seq screen and characterize the promoter elements that facilitate PSPTO_1203-dependent regulation. The genes regulated by PSPTO_1203 encode a TonB-dependent transducer (PSPTO_1206) and a cytoplasmic membrane protein (PSPTO_2145), which is located in the P. syringae pyoverdine cluster. Additionally, we identified siderophores that induce the activity of PSPTO_1203 and used this information to investigate the functional components of the signal transduction cascade.     

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。     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

Shedding light on a Group IV (ECF11) alternative σ factor

This year marks the 50^th anniversary of the discovery of σ⁷⁰ as a protein factor that was needed for bacterial RNA polymerase to accurately transcribe a promoter in vitro. It was 25 years later that the Group IV alternative σs were described as a distinct family of proteins related to σ⁷⁰. In the intervening time, there has been an ever‐growing list of Group IV σs, numbers of genes they transcribe, insight into the diverse suite of processes they control, and appreciation for their impact on bacterial lifestyles. This work summarizes knowledge of the Rhodobacter sphaeroides σ^E‐ChrR pair, a member of the ECF11 subfamily of Group IV alternative σs, in protecting cells from the reactive oxygen species, singlet oxygen. It describes lessons learned from analyzing ChrR, a zinc‐dependent anti‐σ factor, that are generally applicable to Group IV σs and relevant to the response to single oxygen. This MicroReview also illustrates insights into stress responses in this and other bacteria that have been acquired by analyzing or modeling the activity of the σ^E‐ChrR across the bacterial phylogeny.