scholarly journals Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis

2011 ◽  
Vol 78 (5) ◽  
pp. 1593-1595 ◽  
Author(s):  
Chun You ◽  
Xiao-Zhou Zhang ◽  
Y.-H. Percival Zhang
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Restriction Enzyme ◽  
Dna Fragments ◽  
Direct Transformation ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Pcr Product ◽  
Overlap Extension ◽  
General Restriction

ABSTRACTWe developed a general restriction enzyme-free and ligase-free method for subcloning up to three DNA fragments into any location of a plasmid. The DNA multimer generated by prolonged overlap extension PCR was directly transformed inEscherichia coli[e.g., TOP10, DH5α, JM109, and BL21(DE3)] andBacillus subtilisfor obtaining chimeric plasmids.

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 Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

1982 ◽  
Vol 152 (1) ◽  
pp. 275-283
Author(s):  
J Van Randen ◽  
K Wiersma ◽  
G Venema
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gradient Centrifugation ◽  
Cross Linking ◽  
Dna Fragments ◽  
Dna Complexes ◽  
Cscl Gradient ◽  
Donor Moiety

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.

scholarly journals Cellular Stoichiometry of Chemotaxis Proteins in Sinorhizobium meliloti

2020 ◽  
Vol 202 (14) ◽  
Author(s):  
Timofey D. Arapov ◽  
Rafael Castañeda Saldaña ◽  
Amanda L. Sebastian ◽  
W. Keith Ray ◽  
Richard F. Helm ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Sinorhizobium Meliloti ◽  
Response Regulator ◽  
Bacterial Chemotaxis ◽  
Adaptor Proteins ◽  
Molar Ratio ◽  
Content Type ◽  
Chemotaxis Proteins ◽  
Chemotaxis System

ABSTRACT Chemotaxis systems enable microbes to sense their immediate environment, moving toward beneficial stimuli and away from those that are harmful. In an effort to better understand the chemotaxis system of Sinorhizobium meliloti, a symbiont of the legume alfalfa, the cellular stoichiometries of all ten chemotaxis proteins in S. meliloti were determined. A combination of quantitative immunoblot and mass spectrometry revealed that the protein stoichiometries in S. meliloti varied greatly from those in Escherichia coli and Bacillus subtilis. To compare protein ratios to other systems, values were normalized to the central kinase CheA. All S. meliloti chemotaxis proteins exhibited increased ratios to various degrees. The 10-fold higher molar ratio of adaptor proteins CheW1 and CheW2 to CheA might result in the formation of rings in the chemotaxis array that consist of only CheW instead of CheA and CheW in a 1:1 ratio. We hypothesize that the higher ratio of CheA to the main response regulator CheY2 is a consequence of the speed-variable motor in S. meliloti, instead of a switch-type motor. Similarly, proteins involved in signal termination are far more abundant in S. meliloti, which utilizes a phosphate sink mechanism based on CheA retrophosphorylation to inactivate the motor response regulator versus CheZ-catalyzed dephosphorylation as in E. coli and B. subtilis. Finally, the abundance of CheB and CheR, which regulate chemoreceptor methylation, was increased compared to CheA, indicative of variations in the adaptation system of S. meliloti. Collectively, these results mark significant differences in the composition of bacterial chemotaxis systems. IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti contributes greatly to host-plant growth by fixing atmospheric nitrogen. The provision of nitrogen as ammonium by S. meliloti leads to increased biomass production of its legume host alfalfa and diminishes the use of environmentally harmful chemical fertilizers. To better understand the role of chemotaxis in host-microbe interaction, a comprehensive catalogue of the bacterial chemotaxis system is vital, including its composition, function, and regulation. The stoichiometry of chemotaxis proteins in S. meliloti has very few similarities to the systems in Escherichia coli and Bacillus subtilis. In addition, total amounts of proteins are significantly lower. S. meliloti exhibits a chemotaxis system distinct from known models by incorporating new proteins as exemplified by the phosphate sink mechanism.

scholarly journals Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Cierra A. Birch ◽  
Madison J. Davis ◽  
Lea Mbengi ◽  
Peter Zuber
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Α Subunit ◽  
Stress Induction ◽  
Content Type ◽  
Terminal Domain ◽  
Codon Substitution

ABSTRACT Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP α subunit (αCTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in αCTD severely impaired Spx-activated transcription. Attempts to substitute alanine for αCTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where “Y263C” indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound αCTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli αD259, which has been implicated in αCTD-σ70 interaction (σ70 R603, corresponding to R362 of B. subtilis σA). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while αCTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-αCTD complex. IMPORTANCE Though extensively studied in Escherichia coli, the role of αCTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal αCTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-αCTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several αCTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

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 A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Prem P. Kandel ◽  
Hongyu Chen ◽  
Leonardo De La Fuente
Keyword(s):  
Natural Transformation ◽  
Gene Knockout ◽  
Xylella Fastidiosa ◽  
Twitching Motility ◽  
Wild Type ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Type Iv ◽  
Overlap Extension ◽  
Pilin Subunit

ABSTRACT Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2. IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

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 Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

2015 ◽  
Vol 81 (10) ◽  
pp. 3357-3368 ◽  
Author(s):  
Man Hwan Oh ◽  
Je Chul Lee ◽  
Jungmin Kim ◽  
Chul Hee Choi ◽  
Kyudong Han
Keyword(s):  
Antibiotic Resistance ◽  
Acinetobacter Baumannii ◽  
Gene Deletion ◽  
Multidrug Resistant ◽  
Simple Method ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Restriction Sites ◽  
Selection Of

ABSTRACTThe traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistantAcinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection ofA. baumanniimerodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion inA. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition byA. baumanniiATCC 19606 and toompAgene deletion in otherA. baumanniistrains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistantA. baumannii.

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.

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