scholarly journals Distinct clpP Genes Control Specific Adaptive Responses in Bacillus thuringiensis

2002 ◽  
Vol 184 (20) ◽  
pp. 5554-5562 ◽  
Author(s):  
Sinda Fedhila ◽  
Tarek Msadek ◽  
Patricia Nel ◽  
Didier Lereclus
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Low Temperature ◽  
Bacterial Species ◽  
Growth Cycle ◽  
Amino Acid Sequences ◽  
Adaptive Responses ◽  
Regulatory Pathways ◽  
Eukaryotic Organisms ◽  
Mutant Cells

ABSTRACT ClpP and ClpC are subunits of the Clp ATP-dependent protease, which is ubiquitous among prokaryotic and eukaryotic organisms. The role of these proteins in stress tolerance, stationary-phase adaptive responses, and virulence in many bacterial species has been demonstrated. Based on the amino acid sequences of the Bacillus subtilis clpC and clpP genes, we identified one clpC gene and two clpP genes (designated clpP1 and clpP2) in Bacillus thuringiensis. Predicted proteins ClpP1 and ClpP2 have approximately 88 and 67% amino acid sequence identity with ClpP of B. subtilis, respectively. Inactivation of clpC in B. thuringiensis impaired sporulation efficiency. The clpP1 and clpP2 mutants were both slightly susceptible to salt stress, whereas disruption of clpP2 negatively affected sporulation and abolished motility. Virulence of the clp mutants was assessed by injecting bacteria into the hemocoel of Bombyx mori larvae. The clpP1 mutant displayed attenuated virulence, which appeared to be related to its inability to grow at low temperature (25°C), suggesting an essential role for ClpP1 in tolerance of low temperature. Microscopic examination of clpP1 mutant cells grown at 25°C showed altered bacterial division, with cells remaining attached after septum formation. Analysis of lacZ transcriptional fusions showed that clpP1 was expressed at 25 and 37°C during the entire growth cycle. In contrast, clpP2 was expressed at 37°C but not at 25°C, suggesting that ClpP2 cannot compensate for the absence of ClpP1 in the clpP1 mutant cells at low temperature. Our study demonstrates that ClpP1 and ClpP2 control distinct cellular regulatory pathways in B. thuringiensis.

scholarly journals Different Responses of Cytoplasmic and Endoplasmic Reticulum Hsp90 Genes from Eogystia hippophaecola (Lepidoptera: Cossidae) to Cold Stress

Forests ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 1039
Author(s):  
Qianqian Wang ◽  
Jing Tao ◽  
Yurong Li ◽  
Yabei Xu ◽  
Xinhai Liu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Low Temperature ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Response Times ◽  
Expression Patterns ◽  
Amino Acid Sequences ◽  
Sea Buckthorn ◽  
Open Reading Frames

Eogystia hippophaecola Hua, Chou, Fang et Chen (Lepidoptera: Cossidae) is an important borer pest of the sea buckthorn forest (Hippophae rhamnoides L.) in China. Its larvae, which are highly cold tolerant, mainly overwinter in sea buckthorn roots. Heat shock proteins (Hsps) are important molecular chaperones that have been linked to cold tolerance in insects. In this study, we cloned the open reading frames (ORFs) of two Hsp90 genes from E. hippophaecola, EhHsp90-1 and EhHsp90-2, and analyzed their expression under cold stress by qRT-PCR. EhHsp90-1 and EhHsp90-2 are 2154 and 2346 bp in length, respectively, encoding 717 and 781 amino acids. The deduced amino acid sequences contain the conserved signature sequences of the Hsp90 family and the C-terminus characteristic sequence of cytoplasmic or endoplasmic reticulum Hsp90 protein. Phylogenetic analysis revealed the amino acid sequences of EhHsp90-1 and EhHsp90-2 were very similar to the corresponding proteins from Lepidoptera. Under various low-temperature treatments lasting 2 h, EhHsp90-1 and EhHsp90-2 exhibited similar expression patterns, increasing first and then decreasing. At −5 °C, EhHsp90-1 was significantly up-regulated after 12 h, whereas EhHsp90-2 was up-regulated after just 1 h and reached its highest level at 2 h; however, the overall degree of upregulation was greater for EhHsp90-1. Subsequently, the expression level of EhHsp90-2 fluctuated with time. Our results suggest that the two Hsp90s play important roles in E. hippophaecola larvae response to cold stress, but that their response times and the magnitudes of their responses to low-temperature stress differed significantly, providing a theoretical basis for further studying the molecular mechanism of cold tolerance in E. hippophaecola larvae.

scholarly journals Presence of an isoform of H+-pyrophosphatase located in the alveolar sacs of a scuticociliate parasite of turbot: physiological consequences

Parasitology ◽  
2016 ◽  
Vol 143 (5) ◽  
pp. 576-587 ◽  
Author(s):  
NATALIA MALLO ◽  
JESÚS LAMAS ◽  
ANA-PAULA DEFELIPE ◽  
MARIA-EUGENIA DECASTRO ◽  
ROSA-ANA SUEIRO ◽  
...  
Keyword(s):  
Amino Acid ◽  
Physiological Role ◽  
Amino Acid Sequences ◽  
Cell Structures ◽  
Gene And Protein Expression ◽  
Conserved Gene ◽  
Eukaryotic Organisms ◽  
Cellular Compartments ◽  
First Time

SUMMARYH+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasitePhilasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that theP. dicentrarchiH+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance inP. dicentrarchi.

scholarly journals Identification of Listeria monocytogenes Genes Expressed in Response to Growth at Low Temperature

2002 ◽  
Vol 68 (4) ◽  
pp. 1697-1705 ◽  
Author(s):  
Siqing Liu ◽  
James E. Graham ◽  
Lance Bigelow ◽  
Philip D. Morse ◽  
Brian J. Wilkinson
Keyword(s):  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Low Temperature ◽  
Cell Surface ◽  
Stress Responses ◽  
Bacterial Pathogen ◽  
Open Reading Frames ◽  
Cdna Clones ◽  
Adaptive Responses ◽  
Significant Similarity

ABSTRACT Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10°C compared to those grown at 37°C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10°C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.

scholarly journals Sequence Diversity of Bacillus thuringiensis Flagellin (H Antigen) Protein at the Intra-H Serotype Level

2008 ◽  
Vol 74 (17) ◽  
pp. 5524-5532 ◽  
Author(s):  
Dong Xu ◽  
Jean-Charles Côté
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Variable Region ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Sequence Diversity ◽  
Neighbor Joining ◽  
Signature Sequences ◽  
Short Signature ◽  
H Serotypes

ABSTRACT In Bacillus thuringiensis, the hag gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Specific flagellin amino acid sequences have been correlated to specific B. thuringiensis H serotypes, H1 to H67. Ten H serotypes, however, contain three or more antigenic subfactors, labeled a, b, c, d, or e, and have been subdivided into 23 serovars. In the present study, we set out to analyze the sequence diversity of flagellins among serovars from the same H serotypes. We studied the hag genes in 39 B. thuringiensis strains representing the 23 serovars from the 10 H serotypes mentioned above. A serovar and a biovar from an 11th H serotype were also included. The hag genes were amplified and cloned and their nucleotide sequences were determined and translated into amino acid sequences, or the sequences were retrieved directly from GenBank when available. Strains of the H3 serotype contained two or three copies of the fla gene, an ortholog of the hag gene. Strains of the H6 serotype contained three copies. Strains of all other H serotypes each contained a single copy of the hag gene. Alignments of amino acid sequences from all copies in all strains of the H3 serotype revealed short signature sequences, GGAG and SGG, GPDPDDAVKNLT, and DITTTK, that appeared to be specific to the H3c, H3d, and H3e antigenic subfactors, respectively. Similar short signature sequences, GDIT, AFIK, TSAGKA, and SAPSKG, were revealed for H8b, H8c, H20b, and H20c, respectively. Amino acid sequences in the flagellin central variable region were highly conserved among serovars of the H3, H5, H11, and H20 serotypes and much more divergent among serovars of the H4, H10, H18, H24, and H28 serotypes. Two bootstrapped neighbor-joining trees were respectively generated from the alignments of the amino acid sequences translated from all copies of the hag genes in the B. thuringiensis strains of the H3 and H6 serotypes. Sequence identities and relationships were revealed. A third bootstrapped neighbor-joining tree was generated, this one from the alignment of the flagellin amino acid sequences from all the B. thuringiensis strains in the study. Eight clusters, I to VIII, were revealed. Although most clusters contained strains and serovars from the same H serotype, clusters VII and VIII contained serovars from different H serotypes.

Amino acid sequences from the N-terminal domain of Bacillus thuringiensis , subspecies alesti , δ-endotoxin

FEBS Letters ◽  
1986 ◽  
Vol 198 (2) ◽  
pp. 283-286 ◽  
Author(s):  
G.G. Chestukhina ◽  
S.A. Tyurin ◽  
A.L. Osterman ◽  
O.P. Khodova ◽  
V.M. Stepanov
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Amino Acid Sequences ◽  
Terminal Domain

scholarly journals Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Yuki Fujii ◽  
Shiho Tanaka ◽  
Manami Otsuki ◽  
Yasushi Hoshino ◽  
Chinatsu Morimoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
No Reduction ◽  
Amino Acid Sequences ◽  
Binding Affinities ◽  
Binding Mechanism ◽  
Wild Type ◽  
Critical Amino Acid ◽  
Cry Toxin

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.

scholarly journals Identification of Genes of VSH-1, a Prophage-Like Gene Transfer Agent of Brachyspira hyodysenteriae

2005 ◽  
Vol 187 (17) ◽  
pp. 5885-5892 ◽  
Author(s):  
Eric G. Matson ◽  
M. Greg Thompson ◽  
Samuel B. Humphrey ◽  
Richard L. Zuerner ◽  
Thad B. Stanton
Keyword(s):  
Amino Acid ◽  
Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Bacterial Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bacterial Genome ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Brachyspira Hyodysenteriae ◽  
Gene Transfer Agent

ABSTRACT VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Purified VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A λ clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brachyspira species.

scholarly journals Sequence Diversity of the Bacillus thuringiensis and B. cereus Sensu Lato Flagellin (H Antigen) Protein: Comparison with H Serotype Diversity

2006 ◽  
Vol 72 (7) ◽  
pp. 4653-4662 ◽  
Author(s):  
Dong Xu ◽  
Jean-Charles Côté
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Nucleotide Sequences ◽  
Amino Acid Sequences ◽  
Sequence Diversity ◽  
The Other ◽  
Bacillus Halodurans ◽  
V Region ◽  
H Serotypes

ABSTRACT We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagellin allele nucleotide sequences for 10 additional strains were retrieved from GenBank for a total of 106 Bacillus species and strains used in this study. These included 82 B. thuringiensis strains from 67 H serotypes, 5 B. cereus strains, 3 Bacillus anthracis strains, 3 Bacillus mycoides strains, 11 Bacillus weihenstephanensis strains, 1 Bacillus halodurans strain, and 1 Bacillus subtilis strain. The first 111 and the last 66 amino acids were conserved. They were referred to as the C1 and C2 regions, respectively. The central region, however, was highly variable and is referred to as the V region. Two bootstrapped neighbor-joining trees were generated: a first one from the alignment of the translated amino acid sequences of the amplified internal sequences of the hag alleles and a second one from the alignment of the V region amino acid sequences, respectively. Of the eight clusters revealed in the tree inferred from the entire C1-V-C2 region amino acid sequences, seven were present in corresponding clusters in the tree inferred from the V region amino acid sequences. With regard to B. thuringiensis, in most cases, different serovars had different flagellin amino acid sequences, as might have been expected. Surprisingly, however, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. Likewise, serovars from the same H serotypes were most often found clustered together, with exceptions. Indeed, some serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. Species-wise, B. halodurans, B. subtilis, and B. anthracis formed specific branches, whereas the other four species, all in the B. cereus sensu lato group, B. mycoides, B. weihenstephanensis, B. cereus, and B. thuringiensis, did not form four specific clusters as might have been expected. Rather, strains from any of these four species were placed side by side with strains from the other species. In the B. cereus sensu lato group, B. anthracis excepted, the distribution of strains was not species specific.

Characterization ofBacillus thuringiensissubsp.kurstakistrain S93 effective against the fall armywormSpodoptera frugiperda)

1999 ◽  
Vol 45 (6) ◽  
pp. 464-471 ◽  
Author(s):  
Joseilde O Silva-Werneck ◽  
Marlene T De-Souza ◽  
José MC de S. Dias ◽  
Bergmann M Ribeiro
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Spodoptera Frugiperda ◽  
Fall Armyworm ◽  
Amino Acid Sequences ◽  
Dna Library ◽  
Sds Page ◽  
Total Dna ◽  
Type Gene ◽  
High Degree

A Brazilian strain of Bacillus thuringiensis subsp. kurstaki, designated S93, was analyzed regarding its cry gene and protein contents and activity against the fall armyworm (Spodoptera frugiperda, Smith 1797). Bioassays using lyophilized powders of S93 or HD-1 and third instar larvae of S. frugiperda showed a 12.3-fold lower LC50for the S93 strain when compared with the standard HD-1 strain. The spore-crystal mixture, analyzed by SDS-PAGE, showed two major polypeptides of 130 and 65 kDa, corresponding to Cry1 and Cry2 toxins, respectively. Western blot analysis showed that these proteins were immunologically related to the Cry1A protein from B. thuringiensis subsp. kurstaki HD-73. The polymerase chain reaction technique (PCR) using total DNA from the S93 strain and specific primers showed the presence of cry1Aa, cry1Ab, and cry1Ac genes, and a cry1A-type gene was localized in a plasmid of about 44 MDa. A cry1Ab gene was isolated from a S93 plasmid DNA library and completely sequenced. Computer analysis showed that the gene sequence (GenBank acession number AF059670) is identical to cry1Ab1 and has 91.6 and 85.9% identity with cry1Aa1 and cry1Ac1 genes, respectively. The deduced amino-acid sequence showed a high degree of similarity with the amino-acid sequences of the Cry1Ab1 (100%), Cry1Aa1 (93.8%), and Cry1Ac1 (90.6%) proteins.Key words: Bacillus thuringiensis, Spodoptera frugiperda, biological control, crystal protein, cry genes.

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