The bacterial stressosome:a modular system that has been adapted to control secondary messenger signaling

SummaryThe stressosome complex regulates downstream effectors in response to environmental signals. In Bacillus subtilis, it activates the alternative sigma factor σB, leading to the upregulation of the general stress regulon. Herein, we characterize a stressosome-regulated biochemical pathway in Moorella thermoacetica. We show that the presumed sensor, MtR, and the scaffold, MtS, form a pseudo-icosahedral structure like that observed in B. subtilis. The N-terminal domain of MtR is structurally homologous to B. subtilis RsbR, despite low sequence identity. The affinity of the switch kinase, MtT, for MtS decreases following MtS phosphorylation and not because of structural reorganization. Dephosphorylation of MtS by the PP2C type phosphatase MtX permits the switch kinase to rebind the stressosome to reset the response. We also show that MtT regulates cyclic di-GMP biosynthesis through inhibition of a GG(D/E)EF-type diguanylate cyclase, demonstrating that secondary messenger levels are regulated by the stressosome

Engineered Protein Nano-Compartments for Targeted Enzyme Localization

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

A Tale of Two Reductases: Extending the Bacteriochlorophyll Biosynthetic Pathway in <i>E. coli</i>

<div><p>The creation of a synthetic microbe that can harvest energy from sunlight to drive its metabolic processes is an attractive approach to the economically viable biosynthetic production of target compounds. Our aim is to design and engineer a genetically tractable non-photosynthetic microbe to produce light-harvesting molecules. Previously we created a modular, multienzyme system for the heterologous production of intermediates of the bacteriochlorophyll (BChl) pathway in <i>E. coli</i>. In this report we extend this pathway to include a substrate promiscuous 8-vinyl reductase that can accept multiple intermediates of BChl biosynthesis. We present an informative comparative analysis of homologues of 8-vinyl reductase from the model photosynthetic organisms <i>Rhodobacter sphaeroides</i> and <i>Chlorobaculum tepidum</i>. The first purification of the enzymes leads to their detailed biochemical and biophysical characterization. The data obtained reveal that the two 8-vinyl reductases are substrate promiscuous, capable of reducing the C8-vinyl group of Mg protoporphyrin IX, Mg protoporphyrin IX methylester, and divinyl protochlorophyllide. However, activity is dependent upon the presence of chelated Mg²⁺ in the porphyrin ring, with no activity against non-Mg²⁺ chelated intermediates observed. Additionally, CD analyses reveal that the two 8-vinyl reductases appear to bind the same substrate in a different fashion. Furthermore, we discover that the different rates of reaction of the two 8-vinyl reductases both <i>in vitro</i>, and <i>in vivo</i> as part of our engineered system, results in the suitability of only one of the homologues for our BChl pathway in <i>E. coli</i>. Our results offer the first insights into the different functionalities of homologous 8-vinyl reductases. This study also takes us one step closer to the creation of a nonphotosynthetic microbe that is capable of harvesting energy from sunlight for the biosynthesis of molecules of choice.</p></div

Substrate promiscuity of purified 8-vinyl reductases with BChl intermediates as determined by shifts in absorbance maxima.

<p>Conversion of a mixture of Bchl intermediates (MgP^IX, MgP^IXME, P^IXME) was analyzed by HPLC at a single wavelength (412 nm) to detect all porphyrins present in the reaction mixtures after 18 hours. Reactions with enzyme (dotted traces) and control reactions (solid traces) are shown. Wavelengths displayed above arrows (pointing to peak shoulder or peak maximum) indicate the absorbance maximum measured at that time point, and illustrate the 5 nm absorbance shift which occurs after the reduction of the C-8 vinyl group. (<b>A</b>) Purified <i>RS</i>BciA partially reduces the C8-vinyl group of MgP^IX and MgP^IXME to generate a peak shoulder for each substrate at which the absorbance maximum is shifted from 415 nm to 410 nm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Chew2" target="_blank">[27]</a>. Non-Mg chelated compounds are not reduced. Note that the shift in retention time observed for P^IXME in the enzyme and control reaction is the results from an aberrance in column running conditions as both compounds retain the absorbance maximum of the P^IXME substrate. (<b>B</b>) Purified <i>CT</i>BciA reduces the C8-vinyl group on MgP^IX and MgP^IXME, as indicated by a complete shift in compound peak absorbance maxima from 415 nm to 410 nm. No activity and correspondingly, no shift in absorbance maximum is observed against non-Mg chelated compounds P^IX and P^IXME. For abbreviations of substrate names see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone-0089734-t001" target="_blank">Table 1</a>.</p

Bacteriochlorophyll pathway intermediates produced by <i>E. coli</i> cells expressing various combinations of genes from the Heme and BChl pathways as detected by HPLC.

<p><i>E. coli</i> cells expressing HemA-F and the magnesium chelatase complex BchSID produce P^IX and MgP^IX. Addition of the methyl transferase BchM results in production of both P^IX ME and MgP^IX ME. Expression with the divinyl reductase <i>CT</i>BciA in the presence and absence of BchM leads to the production of mono-vinyl forms of pathway intermediates. <i>RS</i>BciA is not active in our <i>in vivo</i> system. Abbreviations: P^IX - protoporphyrin IX, MgP^IX - Mg-protoporphyrin IX, P^IXME - protoporphyrin IX methylester, MgP^IXME - Mg-protoporphyrin IX methylester, mvP^IX - mono-vinyl protoporphyrin IX, mvMgPIX - mono-vinyl Mg-protoporphyrin IX, mvP^IXME - mono-vinyl protoporphyrin IX methylester, mvMgP^IXME - mono-vinyl Mg-protoporphyrin IX methylester, ND – none detected.</p

Reaction efficiency as a measure of percent conversion of divinyl-protochlorophyllide to mono-vinyl protochlorophyllide by 8-vinyl reductase.

<p>Purified <i>CT</i>BciA reduces greater than 85% DVP to mono-vinyl form in 1.5 hours (black bar). Purified <i>RS</i>BciA acts more slowly, reaching 100% conversion of divinyl to mono-vinyl in 18 hours (hashed bars). Attempts to improve reaction efficiency of <i>RS</i>BciA by addition of crude cell lysate to the reaction vessel actually reduced the rate of reaction as well as the overall conversion to less than 80% in 18 hours (white bars). Error bars are calculated from reactions carried out in duplicate.</p

Engineered pathway design for the heterologous production of BChl in the non-photosynthetic host <i>E. coli</i>.

<p>Using succinyl-CoA and glycine as precursor molecules, expression of the heme pathway enzymes HemA-F in <i>E. coli</i> results in production of P^IX as the common intermediate of the heme and BChl biosynthetic pathways. Addition of the BChl enzymes magnesium chelatase (BchHID) and methyltransferase (BchM) yields MgP^IX and MgP^IXME in <i>E. coli </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Kwon1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Johnson3" target="_blank">[15]</a>. Subsequent steps have not yet been functionally assembled in a heterologous system and depending on the enzymes substrate specificities, the order in which the enzymes operate may differ from the depicted pathway. Briefly, formation of the characteristic fifth E ring of chlorophylls is catalyzed by two unrelated and yet to be biochemically characterized cyclases AcsF (aerobic) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Tang1" target="_blank">[20]</a> or BchE (anaerobic) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-BoldarevaNuianzina1" target="_blank">[19]</a>. The D pyrrole ring is reduced either by a light-dependent, nitrogenase-like (LPOR, three-subunit enzyme BchLNB) or a light-independent (DPOR) protochlorophyllide reductase; both enzymes have been biochemically characterized <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Brocker1" target="_blank">[23]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Paddock1" target="_blank">[26]</a>. Reduction of the C8-vinyl group of BChl intermediates is catalyzed by the NADPH-dependent reductase BciA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Chew2" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Canniffe1" target="_blank">[30]</a> investigated in this study. Seven additional enzymatic steps are required for production of Bchl <i>a </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089734#pone.0089734-Willows1" target="_blank">[14]</a>.</p

Localization of EutC^1–19-EGFP in recombinant <i>E. coli</i> expressing <i>S. enterica</i> Eut shell proteins.

<p>Fluorescence microscopy images of <i>E. coli</i> C2566 cells co-expressing EGFP or EutC^1–19-EGFP with EutS (wild type or the G39V mutant), EutMNLK or EutSMNLK. Cell boundaries are shown by the DIC images. (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342.s013" target="_blank">Table S2</a></b> for the quantification of EGFP localization in recombinant <i>E. coli</i>, and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342.s004" target="_blank">Fig. S4</a></b> for the localization of EutC^1–19-EGFP in the <i>E. coli</i> JM109 strain).</p

Hydrolysis of X-gal by <i>E. coli</i> co-expressing EutC^1–19-β-galactosidase and recombinant Eut shell proteins.

<p><i>E. coli</i> C2566 cells with constructs for constitutive expression of β-galactosidase (β-gal) or EutC^1–19-β-gal and different combinations of Eut shell proteins were grown with the β-gal substrate X-gal. Intracellular accumulation of the insoluble X-gal cleavage product was observed by Differential Interference Contrast (DIC) microscopy. Arrows point to intracellular indole deposits.</p