scholarly journals Cytoplasmic pH Response to Acid Stress in Individual Cells of Escherichia coli and Bacillus subtilis Observed by Fluorescence Ratio Imaging Microscopy

2012 ◽  
Vol 78 (10) ◽  
pp. 3706-3714 ◽  
Author(s):  
Keith A. Martinez ◽  
Ryan D. Kitko ◽  
J. Patrick Mershon ◽  
Haley E. Adcox ◽  
Kotiba A. Malek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Acid Stress ◽  
Adherent Cells ◽  
Cytoplasmic Ph ◽  
Fluorescence Ratio ◽  
Isolated Cells ◽  
Content Type ◽  
E Coli ◽  
Standard Curve

ABSTRACTThe ability ofEscherichia coliandBacillus subtilisto regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. TheE. colicytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In anE. colibiofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells.B. subtiliscells were acid shifted from pH 7.5 to pH 6.0. InB. subtilis, unlike the case withE. coli, cytoplasmic pH showed no “overshoot” but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individualB. subtiliscells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.

scholarly journals Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Charles T. Lauhon
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genomic Context ◽  
Similar Reduction ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Fertile Ground ◽  
And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

scholarly journals Escherichia coli YqjA, a Member of the Conserved DedA/Tvp38 Membrane Protein Family, Is a Putative Osmosensing Transporter Required for Growth at Alkaline pH

2015 ◽  
Vol 197 (14) ◽  
pp. 2292-2300 ◽  
Author(s):  
Sujeet Kumar ◽  
William T. Doerrler
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Amino Acid Identity ◽  
Protein Family ◽  
Cytoplasmic Ph ◽  
Alkaline Ph ◽  
Ph Homeostasis ◽  
Content Type ◽  
E Coli ◽  
Alkaline Conditions

ABSTRACTThe ability to persist and grow under alkaline conditions is an important characteristic of many bacteria. In order to survive at alkaline pH,Escherichia colimust maintain a stable cytoplasmic pH of about 7.6. Membrane cation/proton antiporters play a major role in alkaline pH homeostasis by catalyzing active inward proton transport. The DedA/Tvp38 family is a highly conserved membrane protein family of unknown function present in most sequenced genomes. YqjA and YghB are members of theE. coliDedA family with 62% amino acid identity and partially redundant functions. We have shown thatE. coliwith ΔyqjAand ΔyghBmutations cannot properly maintain the proton motive force (PMF) and is compromised in PMF-dependent drug efflux and other PMF-dependent functions. Furthermore, the functions of YqjA and YghB are dependent upon membrane-embedded acidic amino acids, a hallmark of several families of proton-dependent transporters. Here, we show that the ΔyqjAmutant (but not ΔyghB) cannot grow under alkaline conditions (ranging from pH 8.5 to 9.5), unlike the parentE. coli. Overexpression ofyqjArestores growth at alkaline pH, but only when more than ∼100 mM sodium or potassium is present in the growth medium. Increasing the osmotic pressure by the addition of sucrose enhances the ability of YqjA to support growth under alkaline conditions in the presence of low salt concentrations, consistent with YqjA functioning as an osmosensor. We suggest that YqjA possesses proton-dependent transport activity that is stimulated by osmolarity and that it plays a significant role in the survival ofE. coliat alkaline pH.IMPORTANCEThe ability to survive under alkaline conditions is important for many species of bacteria.Escherichia colican grow at pH 5.5 to 9.5 while maintaining a constant cytoplasmic pH of about 7.6. Under alkaline conditions, bacteria rely upon proton-dependent transporters to maintain a constant cytoplasmic pH. The DedA/Tvp38 protein family is a highly conserved but poorly characterized family of membrane proteins. Here, we show that the DedA/Tvp38 protein YqjA is critical forE. colito survive at pH 8.5 to 9.5. YqjA requires sodium and potassium for this function. At low cation concentrations, osmolytes, including sucrose, can facilitate rescue ofE. coligrowth by YqjA at high pH. These data are consistent with YqjA functioning as an osmosensing cation-dependent proton transporter.

scholarly journals Pirated Siderophores Promote Sporulation in Bacillus subtilis

2017 ◽  
Vol 83 (10) ◽  
Author(s):  
Gabrielle M. Grandchamp ◽  
Lews Caro ◽  
Elizabeth A. Shank
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Iron Acquisition ◽  
Microbial Interactions ◽  
Mineral Nutrient ◽  
Cell Physiology ◽  
Content Type ◽  
E Coli ◽  
Esterase Gene

ABSTRACT In microbial communities, bacteria chemically and physically interact with one another. Some of these interactions are mediated by secreted specialized metabolites that act as either intraspecies or interspecies signals to alter gene expression and to change cell physiology. Bacillus subtilis is a well-characterized soil microbe that can differentiate into multiple cell types, including metabolically dormant endospores. We were interested in identifying microbial interactions that affected sporulation in B. subtilis. Using a fluorescent transcriptional reporter, we observed that coculturing B. subtilis with Escherichia coli promoted sporulation gene expression via a secreted metabolite. To identify the active compound, we screened the E. coli Keio Collection and identified the sporulation-accelerating cue as the siderophore enterobactin. B. subtilis has multiple iron acquisition systems that are used to take up the B. subtilis-produced siderophore bacillibactin, as well as to pirate exogenous siderophores such as enterobactin. While B. subtilis uses a single substrate binding protein (FeuA) to take up both bacillibactin and enterobactin, we discovered that it requires two distinct genes to sporulate in response to these siderophores (the esterase gene besA for bacillibactin and a putative esterase gene, ybbA, for enterobactin). In addition, we found that siderophores from a variety of other microbial species also promote sporulation in B. subtilis. Our results thus demonstrate that siderophores can act not only as bacterial iron acquisition systems but also as interspecies cues that alter cellular development and accelerate sporulation in B. subtilis. IMPORTANCE While much is known about the genetic regulation of Bacillus subtilis sporulation, little is understood about how other bacteria influence this process. This work describes an interaction between Escherichia coli and B. subtilis that accelerates sporulation in B. subtilis. The interaction is mediated by the E. coli siderophore enterobactin; we show that other species' siderophores also promote sporulation gene expression in B. subtilis. These results suggest that siderophores not only may supply bacteria with the mineral nutrient iron but also may play a role in bacterial interspecies signaling, providing a cue for sporulation. Siderophores are produced by many bacterial species and thus potentially play important roles in altering bacterial cell physiology in diverse environments.

scholarly journals Distinct Transcriptional Profiles and Phenotypes Exhibited by Escherichia coli O157:H7 Isolates Related to the 2006 Spinach-Associated Outbreak

2011 ◽  
Vol 78 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Craig T. Parker ◽  
Jennifer L. Kyle ◽  
Steven Huynh ◽  
Michelle Q. Carter ◽  
Maria T. Brandl ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Stress ◽  
The United States ◽  
Clinical Isolates ◽  
Content Type ◽  
Transcriptional Profiles ◽  
E Coli ◽  
Large Outbreak ◽  
Altered Gene ◽  
Feral Pig

ABSTRACTIn 2006, a large outbreak ofEscherichia coliO157:H7 was linked to the consumption of ready-to-eat bagged baby spinach in the United States. The likely sources of preharvest spinach contamination were soil and water that became contaminated via cattle or feral pigs in the proximity of the spinach fields. In this study, we compared the transcriptional profiles of 12E. coliO157:H7 isolates that possess the same two-enzyme pulsed-field gel electrophoresis (PFGE) profile and are related temporally or geographically to the above outbreak. TheseE. coliO157:H7 isolates included three clinical isolates, five isolates from separate bags of spinach, and single isolates from pasture soil, river water, cow feces, and a feral pig. The three clinical isolates and two spinach bag isolates grown in cultures to stationary phase showed decreased expression of many σS-regulated genes, includinggadA,osmE,osmY, andkatE, compared with the soil, water, cow, feral pig, and the other three spinach bag isolates. The decreased expression of these σS-regulated genes was correlated with the decreased resistance of the isolates to acid stress, osmotic stress, and oxidative stress but increases in scavenging ability. We also observed that intraisolate variability was much more pronounced among the clinical and spinach isolates than among the environmental isolates. Together, the transcriptional and phenotypic differences of the spinach outbreak isolates ofE. coliO157:H7 support the hypothesis that some variants within the spinach bag retained characteristics of the preharvest isolates, whereas other variants with altered gene expression and phenotypes infected the human host.

scholarly journals Cellular Stoichiometry of Methyl-Accepting Chemotaxis Proteins inSinorhizobium meliloti

2017 ◽  
Vol 200 (6) ◽  
Author(s):  
Hardik M. Zatakia ◽  
Timofey D. Arapov ◽  
Veronika M. Meier ◽  
Birgit E. Scharf
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Sinorhizobium Meliloti ◽  
High Abundance ◽  
Detailed Knowledge ◽  
Class Iii ◽  
Content Type ◽  
E Coli ◽  
Chemotaxis Proteins ◽  
Carboxy Terminal

ABSTRACTThe chemosensory system inSinorhizobium melilotihas several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA inS. meliloti. Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell inS. meliloti. The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen inBacillus subtilis(23:1) and about 10 times higher than that inEscherichia coli(3.4:1). Different fromE. coli, the high-abundance receptors inS. melilotiare lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptionallacZfusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system inS. melilotideviates significantly from the established enterobacterial paradigm but shares some similarities withB. subtilis.IMPORTANCEThe symbiotic soil bacteriumSinorhizobium melilotiis of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better understanding of the role of chemotaxis in these processes, detailed knowledge on the regulation and composition of the chemosensory machinery is essential. Here, we show that chemoreceptor gene expression inS. melilotiis controlled through the main transcriptional regulators of motility. Chemoreceptor abundance is much lower inS. melilotithan inEscherichia coliandBacillus subtilis. Moreover, the chemoreceptor-to-kinase CheA ratio is different from that ofE. colibut similar to that ofB. subtilis.

scholarly journals Acetoin Synthesis Acquisition Favors Escherichia coli Growth at Low pH

2014 ◽  
Vol 80 (19) ◽  
pp. 6054-6061 ◽  
Author(s):  
Bram Vivijs ◽  
Pieter Moons ◽  
Abram Aertsen ◽  
Chris W. Michiels
Keyword(s):  
Escherichia Coli ◽  
Culture Media ◽  
Acid Stress ◽  
Low Ph ◽  
Phase Cell ◽  
Acid Fermentation ◽  
Serratia Plymuthica ◽  
Content Type ◽  
E Coli ◽  
Acetoin Production

ABSTRACTSome members of the familyEnterobacteriaceaeferment sugars via the mixed-acid fermentation pathway. This yields large amounts of acids, causing strong and sometimes even lethal acidification of the environment. Other family members employ the 2,3-butanediol fermentation pathway, which generates comparatively less acidic and more neutral end products, such as acetoin and 2,3-butanediol. In this work, we equippedEscherichia coliMG1655 with thebudABoperon, encoding the acetoin pathway, fromSerratia plymuthicaRVH1 and investigated how this affected the ability ofE. colito cope with acid stress during growth. Acetoin fermentation prevented lethal medium acidification byE. coliin lysogeny broth (LB) supplemented with glucose. It also supported growth and higher stationary-phase cell densities in acidified LB broth with glucose (pH 4.10 to 4.50) and in tomato juice (pH 4.40 to 5.00) and reduced the minimal pH at which growth could be initiated. On the other hand, the acetoin-producing strain was outcompeted by the nonproducer in a mixed-culture experiment at low pH, suggesting a fitness cost associated with acetoin production. Finally, we showed that acetoin production profoundly changes the appearance ofE. colion several diagnostic culture media. NaturalE. colistrains that have laterally acquiredbudABgenes may therefore have escaped detection thus far. This study demonstrates the potential importance of acetoin fermentation in the ecology ofE. coliin the food chain and contributes to a better understanding of the microbiological stability and safety of acidic foods.

scholarly journals Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

2015 ◽  
Vol 197 (11) ◽  
pp. 1952-1962 ◽  
Author(s):  
Katherine A. Black ◽  
Patricia C. Dos Santos
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Lysine Trna ◽  
Domains Of Life

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

scholarly journals Acid-Adapted Strains of Escherichia coli K-12 Obtained by Experimental Evolution

2015 ◽  
Vol 81 (6) ◽  
pp. 1932-1941 ◽  
Author(s):  
Mark M. Harden ◽  
Amanda He ◽  
Kaitlin Creamer ◽  
Michelle W. Clark ◽  
Issam Hamdallah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Acid Stress ◽  
Enteric Bacteria ◽  
Missense Mutations ◽  
Cytoplasmic Ph ◽  
Content Type ◽  
Fitness Advantage ◽  
K 12 ◽  
External Ph

ABSTRACTEnteric bacteria encounter a wide range of pHs throughout the human intestinal tract. We conducted experimental evolution ofEscherichia coliK-12 to isolate clones with increased fitness during growth under acidic conditions (pH 4.5 to 4.8). Twenty-four independent populations ofE. coliK-12 W3110 were evolved in LBK medium (10 g/liter tryptone, 5 g/liter yeast extract, 7.45 g/liter KCl) buffered with homopiperazine-N,N′-bis-2-(ethanosulfonic acid) and malate at pH 4.8. At generation 730, the pH was decreased to 4.6 with HCl. By 2,000 generations, all populations had achieved higher endpoint growth than the ancestor at pH 4.6 but not at pH 7.0. All evolving populations showed a progressive loss of activity of lysine decarboxylase (CadA), a major acid stress enzyme. This finding suggests a surprising association between acid adaptation and moderation of an acid stress response. At generation 2,000, eight clones were isolated from four populations, and their genomes were sequenced. Each clone showed between three and eight missense mutations, including one in a subunit of the RNA polymerase holoenzyme (rpoB,rpoC, orrpoD). Missense mutations were found inadiY, the activator of the acid-inducible arginine decarboxylase (adiA), and ingcvP(glycine decarboxylase), a possible acid stress component. For tests of fitness relative to that of the ancestor,lacZ::kanwas transduced into each strain. All acid-evolved clones showed a high fitness advantage at pH 4.6. With the cytoplasmic pH depressed by benzoate (at external pH 6.5), acid-evolved clones showed decreased fitness; thus, there was no adaptation to cytoplasmic pH depression. At pH 9.0, acid-evolved clones showed no fitness advantage. Thus, our acid-evolved clones showed a fitness increase specific to low external pH.

scholarly journals Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Daniel Parrell ◽  
Yang Zhang ◽  
Sandra Olenic ◽  
Lee Kroos
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Membrane Proteins ◽  
Direct Effects ◽  
Transmembrane Segment ◽  
Content Type ◽  
E Coli ◽  
Transmembrane Segments

ABSTRACT RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli, we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli. Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli, including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His6-MBP-RsiW(73–118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His6-MBP-FtsL(23–117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro. We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli, providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro, suggesting that FtsL has an additional requirement.

scholarly journals A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Miglena Manandhar ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Chemical Synthesis ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Bacillus Sphaericus ◽  
Content Type ◽  
E Coli

ABSTRACTBioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed,in vitrotheEscherichia coliandBacillus sphaericusenzymes have been shown to condense pimeloyl-CoA withl-alanine in a pyridoxal 5′-phosphate-dependent reaction with concomitant CoA release and decarboxylation ofl-alanine. However, recentin vivostudies ofE. coliandBacillus subtilissuggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in thatE. coliBioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast,B. subtilisBioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic andin vitrodata demonstrating thatB. subtilisBioF specifically utilizes pimeloyl-CoA.IMPORTANCEBiotin is an essential vitamin required by mammals and birds because, unlike bacteria, plants, and some fungi, these organisms cannot make biotin. Currently, the biotin included in vitamin tablets and animal feeds is made by chemical synthesis. This is partly because the biosynthetic pathways in bacteria are incompletely understood. This paper defines an enzyme of theBacillus subtilispathway and shows that it differs from that ofEscherichia coliin the ability to utilize specific precursors. These bacteria have been used in biotin production and these data may aid in making biotin produced by biotechnology commercially competitive with that produced by chemical synthesis.

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