scholarly journals Isolation of RNase H Genes That Are Essential for Growth of Bacillus subtilis 168

1999 ◽  
Vol 181 (7) ◽  
pp. 2118-2123 ◽  
Author(s):  
Mitsuhiro Itaya ◽  
Akira Omori ◽  
Shigenori Kanaya ◽  
Robert J. Crouch ◽  
Teruo Tanaka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rnase H ◽  
Positive Bacterium ◽  
E Coli ◽  
Cellular Processes ◽  
Dna Library ◽  
Bacillus Subtilis 168 ◽  
Genes Encoding

ABSTRACT Two genes encoding functional RNase H (EC 3.1.26.4 ) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilisRNase HIII. The B. subtilis genome has anrnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB andrnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both thernhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the singlernhC homologue in the small genome ofMycoplasma species.

scholarly journals Preventive antimicrobial action and tissue architecture ameliorations of Bacillus subtilis in challenged broilers

2021 ◽  
Vol 14 (2) ◽  
pp. 523-536
Author(s):  
Essam S. Soliman ◽  
Rania T. Hamad ◽  
Mona S. Abdallah
Keyword(s):  
Bacillus Subtilis ◽  
Broiler Chickens ◽  
Trichophyton Mentagrophytes ◽  
Antimicrobial Activities ◽  
Lymphoid Hyperplasia ◽  
Bursa Of Fabricius ◽  
Tissue Architecture ◽  
E Coli

Background and Aim: Probiotics improve intestinal balance through bacterial antagonism and competitive exclusion. This study aimed to investigate the in vitro antimicrobial activity, as well as the in vivo preventive, immunological, productive, and histopathological modifications produced by probiotic Bacillus subtilis. Materials and Methods: The in vitro antimicrobial activities of B. subtilis (5×106 CFU/g; 0.5, 1.0*, 1.5, and 2.0 g/L) were tested against Escherichia coli O157: H7, Salmonella Typhimurium, Candida albicans, and Trichophyton mentagrophytes after exposure times of 0.25, 0.5, 1, and 2 h using minimal inhibitory concentration procedures. A total of 320 1-day-old female Ross broiler chickens were divided into five groups. Four out of the five groups were supplemented with 0.5, 1.0*, 1.5, and 2.0 g/L probiotic B. subtilis from the age of 1 day old. Supplemented 14-day-old broiler chickens were challenged with only E. coli O157: H7 (4.5×1012 CFU/mL) and S. Typhimurium (1.2×107 CFU/mL). A total of 2461 samples (256 microbial-probiotic mixtures, 315 sera, 315 duodenal swabs, and 1575 organs) were collected. Results: The in vitro results revealed highly significant (p<0.001) killing rates at all-time points in 2.0 g/L B. subtilis: 99.9%, 90.0%, 95.6%, and 98.8% against E. coli, S. Typhimurium, C. albicans, and T. mentagrophytes, respectively. Broilers supplemented with 1.5 and 2.0 g/L B. subtilis revealed highly significant increases (p<0.01) in body weights, weight gains, carcass weights, edible organs' weights, immune organs' weights, biochemical profile, and immunoglobulin concentrations, as well as highly significant declines (p<0.01) in total bacterial, Enterobacteriaceae, and Salmonella counts. Histopathological photomicrographs revealed pronounced improvements and near-normal pictures of the livers and hearts of broilers with lymphoid hyperplasia in the bursa of Fabricius, thymus, and spleen after supplementation with 2.0 g/L B. subtilis. Conclusion: The studies revealed that 1.5-2.0 g of probiotic B. subtilis at a concentration of 5×106 CFU/g/L water was able to improve performance, enhance immunity, and tissue architecture, and produce direct antimicrobial actions.

scholarly journals Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

2015 ◽  
Vol 83 (4) ◽  
pp. 1384-1395 ◽  
Author(s):  
Aimee Tan ◽  
Nicola K. Petty ◽  
Dianna Hocking ◽  
Vicki Bennett-Wood ◽  
Matthew Wakefield ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Gene Repertoire ◽  
Citrobacter Rodentium ◽  
Environmental Strain ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogenCitrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and thegrlRAoperon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genusEscherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated thatregAhas been horizontally transferred toEscherichiaspp. andC. rodentium. Comparative studies of two RegA homologues, namely, those fromC. rodentiumandE. coliSMS-3-5, a multiresistant environmental strain ofE. coli, showed that the two regulators acted similarlyin vitrobut differed in terms of their abilities to activate the virulence ofC. rodentiumin vivo, which evidently was due to their differential activation ofgrlRA. Our data indicate that RegA fromC. rodentiumhas strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence byC. rodentiumand on the evolution of virulence-regulatory genes of bacterial pathogens in general.

scholarly journals Interaction of Bacillus subtilis CsaA with SecA and precursor proteins

2000 ◽  
Vol 348 (2) ◽  
pp. 367-373 ◽  
Author(s):  
Jörg P. MÜLLER ◽  
Jörg OZEGOWSKI ◽  
Stefan VETTERMANN ◽  
Jelto SWAVING ◽  
Karel H. M. VAN WELY ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Specific Interaction ◽  
Membrane Vesicles ◽  
Protein Export ◽  
E Coli ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

CsaA from the Gram-positive bacterium Bacillus subtilis has been identified previously as a suppressor of the growth and protein-export defect of Escherichia coli secA(Ts) mutants. CsaA has chaperone-like activities in vivo and in vitro. To examine the role of CsaA in protein export in B. subtilis, expression of the csaA gene was repressed. While export of most proteins remained unaffected, export of at least two proteins was significantly reduced upon CsaA depletion. CsaA co-immunoprecipitates and co-purifies with the SecA proteins of E. coli and B. subtilis, and binds the B. subtilis preprotein prePhoB. Purified CsaA stimulates the translocation of prePhoB into E. coli membrane vesicles bearing the B. subtilis translocase, whereas it interferes with the SecB-mediated translocation of proOmpA into membrane vesicles of E. coli. The specific interaction with the SecA translocation ATPase and preproteins suggests that CsaA acts as a chaperone that promotes the export of a subset of preproteins in B. subtilis.

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.

The ATPase activity of an ‘essential’ Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 944-956 ◽  
Author(s):  
Johanna C. Karst ◽  
Anne-Emmanuelle Foucher ◽  
Tracey L. Campbell ◽  
Anne-Marie Di Guilmi ◽  
David Stroebel ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Atpase Activity ◽  
Normal Growth ◽  
Conditional Knockout ◽  
Protein Oligomerization ◽  
E Coli ◽  
Oligomeric Form ◽  
High Concentrations

Characterization of ‘unknown’ proteins is one of the challenges of the post-genomic era. Here, we report a study of Bacillus subtilis YdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of the ydiB gene yielded a mutant with much reduced growth rate compared to the wild-type strain. In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed a K m of about 60 μM for ATP and a V max of about 10 nmol min−1 (mg protein)−1 (k cat ∼10 h−1). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain for ydiB, showing that the ATPase activity of YdiB is required for the in vivo function of the protein. Oligomerization was also observed with the purified YjeE from Escherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in a B. subtilis extract, or when stabilized by formaldehyde cross-linking for YjeE from E. coli, suggesting that oligomerization might regulate the function of this new class of proteins in vivo.

scholarly journals Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

2021 ◽  
Author(s):  
Devon A. Stork ◽  
Georgia R. Squyres ◽  
Erkin Kuru ◽  
Katarzyna A. Gromek ◽  
Jonathan Rittichier ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Code ◽  
Cell Biology ◽  
Stop Codon ◽  
Positive Bacterium ◽  
E Coli ◽  
Binding Interface ◽  
Genetic Code Expansion ◽  
And Control

AbstractBacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

scholarly journals Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

2006 ◽  
Vol 72 (12) ◽  
pp. 7730-7738 ◽  
Author(s):  
Stephanie Shipkowski ◽  
Jean E. Brenchley
Keyword(s):  
Glycoside Hydrolase ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Type I ◽  
Plant Polysaccharide ◽  
E Coli ◽  
Genes Encoding ◽  
Layer Chromatography

ABSTRACT Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.

scholarly journals Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Devon A. Stork ◽  
Georgia R. Squyres ◽  
Erkin Kuru ◽  
Katarzyna A. Gromek ◽  
Jonathan Rittichier ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Code ◽  
Cell Biology ◽  
Stop Codon ◽  
Positive Bacterium ◽  
E Coli ◽  
Binding Interface ◽  
Genetic Code Expansion ◽  
And Control

AbstractBacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

scholarly journals The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning

1999 ◽  
Vol 181 (19) ◽  
pp. 6053-6062 ◽  
Author(s):  
Stephen A. Sciochetti ◽  
Patrick J. Piggot ◽  
David J. Sherratt ◽  
Garry Blakely
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Holliday Junction ◽  
Independent Manner ◽  
Site Specific ◽  
E Coli ◽  
Chromosome Partitioning ◽  
A Site

ABSTRACT The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers inB. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripXstrains by the first division after the initiation of germination. The introduction of a recA mutation into ripXstrains resulted in only slight modifications of the ripXphenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing adif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.

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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