Genetic and functional analysis of the cytK family of genes in Bacillus cereus

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2689-2697 ◽  
Author(s):  
Annette Fagerlund ◽  
Ola Ween ◽  
Terje Lund ◽  
Simon P. Hardy ◽  
Per E. Granum
Keyword(s):  
Bacillus Cereus ◽  
Lipid Bilayers ◽  
Dna Sequences ◽  
Vero Cells ◽  
Amino Acid Sequences ◽  
Planar Lipid Bilayers ◽  
Necrotic Enteritis ◽  
Related Gene ◽  
Pcr Products ◽  
Very High

CytK is a pore-forming toxin of Bacillus cereus that has been linked to a case of necrotic enteritis. PCR products of the expected size were generated with cytK primers in 13 of 29 strains. Six strains were PCR-positive for the related gene hly-II, which encodes haemolysin II, a protein that is 37 % identical to the original CytK. Five of the strains were positive for both genes. The DNA sequences of putative cytK genes from three positive strains were determined, and the deduced amino acid sequences were 89 % identical to that of the original CytK. The authors have designated this new cytK variant cytK-2, and refer to the original cytK as cytK-1. The CytK-2 proteins from these three strains were isolated, and their identity was verified by N-terminal sequencing. blast analysis using the cytK-2 gene sequences revealed very high homology with two cytK-2 sequences in the genomes of B. cereus strains ATCC 14579 and ATCC 10987. The differences between CytK-1 and the CytK-2 proteins were clustered to certain regions of the proteins. The isolated CytK-2 proteins were haemolytic and toxic towards human intestinal Caco-2 cells and Vero cells, although their toxicity was about 20 % of that of CytK-1. Both native and recombinant CytK-2 proteins from B. cereus 1230-88 were able to form pores in planar lipid bilayers, but the majority of the channels observed were of lower conductance than those created by CytK-1. It is likely that CytK-2 toxins contribute to the enterotoxicity of several strains of B. cereus, although not all of the CytK-2 toxins may be as harmful as the CytK-1 originally isolated.

scholarly journals Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Xu-Xia Yan ◽  
Corrine J. Porter ◽  
Simon P. Hardy ◽  
David Steer ◽  
A. Ian Smith ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Clostridium Perfringens ◽  
Pore Formation ◽  
Random Mutagenesis ◽  
Planar Lipid Bilayers ◽  
Necrotic Enteritis ◽  
Channel Conductance ◽  
Content Type ◽  
Alpha Hemolysin ◽  
Insight Into

ABSTRACT Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

scholarly journals Saturation behavior of single, amiloride-sensitive Na+ channels in planar lipid bilayers

1984 ◽  
Vol 46 (6) ◽  
pp. 831-835 ◽  
Author(s):  
L. Olans ◽  
S. Sariban-Sohraby ◽  
D.J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Na Channels

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

scholarly journals Prevalence of human papillomavirus DNA sequences in an area with very high incidence of cervical carcinoma

1994 ◽  
Vol 70 (4) ◽  
pp. 694-696 ◽  
Author(s):  
CC Pao ◽  
SM Kao ◽  
G-C Tang ◽  
K Lee ◽  
J Si ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cervical Carcinoma ◽  
Dna Sequences ◽  
High Incidence ◽  
Papillomavirus Dna ◽  
Very High

scholarly journals Purification and characterization of a uterine retinol-binding protein in the bitch

1995 ◽  
Vol 311 (2) ◽  
pp. 407-415 ◽  
Author(s):  
W C Buhi ◽  
I M Alvarez ◽  
V M Shille ◽  
M J Thatcher ◽  
J P Harney ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequences ◽  
Binding Protein ◽  
Amino Acid Content ◽  
Amino Acid Sequences ◽  
Retinol Binding Protein ◽  
Total Amino Acid ◽  
Major Protein ◽  
Serum Retinol ◽  
Two Dimensional Gel Electrophoresis

A major canine endometrial secreted protein (cP6, 23,000-M(r)) was purified by ion-exchange and gel-filtration chromatography and characterized by two-dimensional gel electrophoresis. Anti-[human retinol-binding protein (hRBP)] serum identified cP6 on immunoblot analysis and immunoprecipitated cP6 from culture medium. This major protein was also shown to bind [3H]retinol. N-terminal and internal amino acid sequences were determined and compared with previously identified protein, RNA, or DNA sequences. N-terminal analysis revealed that cP6 had high identity and similarity to serum retinol-binding proteins (RBPs), while internal sequence analysis showed a strong similarity to rat androgen-dependent epididymal protein and beta-lactoglobulins. Amino acid analysis, however, showed significant differences between these proteins and cP6 in both total amino acid content and certain selected amino acids. Immunohistochemical analysis showed staining for RBP only in the uterine luminal epithelium. These studies suggest that bitch endometrium secretes a family of proteins (cP6), some of which bind [3H]retinol, are immunologically related to the RBP family, and have N-terminal and internal sequences with a high similarity to RBP, beta-lactoglobulins and other members of the lipocalin family. This family of proteins may be important in early development for supplying retinol or derivatives to the developing embryo.

scholarly journals Stimulation‐dependent gating of TRPM3 channel in planar lipid bilayers

2015 ◽  
Vol 30 (3) ◽  
pp. 1306-1316 ◽  
Author(s):  
Kunitoshi Uchida ◽  
Lusine Demirkhanyan ◽  
Swapna Asuthkar ◽  
Alejandro Cohen ◽  
Makoto Tominaga ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Planar Lipid Bilayers

Multiple calmodulin mRNA species are derived from two distinct genes

1987 ◽  
Vol 7 (5) ◽  
pp. 1873-1880
Author(s):  
H Nojima ◽  
K Kishi ◽  
H Sokabe
Keyword(s):  
Dna Sequences ◽  
Northern Blotting ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Coding Region ◽  
Genomic Libraries ◽  
Mrna Species ◽  
Nuclease Mapping ◽  
Rat Genome

We have observed three calmodulin mRNA species in rat tissues. In order to know from how many expressed genes they are derived, we have investigated the genomic organization of calmodulin genes in the rat genome. From a rat brain cDNA library, we obtained two kinds of cDNAs (pRCM1 and pRCM3) encoding authentic calmodulin. DNA sequence analysis of these cDNA clones revealed substitutions of nucleotides at 73 positions of 450 nucleotides in the coding region, although the amino acid sequences of these calmodulins are exactly the same. DNA sequences in the 5' and 3' noncoding regions are quite different between these two cDNAs. From these results, we conclude that they are derived from two distinct bona fide calmodulin genes, CaMI (pRCM1) and CaMII (pRCM3). Total genomic Southern hybridization suggested four distinct calmodulin-related genes in the rat genome. By cloning and sequencing the calmodulin-related genes from rat genomic libraries, we demonstrated that the other two genes are processed pseudogenes generated from the CaMI (lambda SC9) and CaMII (lambda SC8) genes, respectively, through an mRNA-mediated process of insertions. Northern blotting showed that the CaMI gene is transcribed in liver, muscle, and brain in similar amounts, whereas the CaMII gene is transcribed mainly in brain. S1 nuclease mapping indicated that the CaMI gene produced two mRNA species (1.7 and 4 kilobases), whereas the CaMII gene expressed a single mRNA species (1.4 kilobases).

Structural and antigenic polymorphism of the 35- to 48-kilodalton merozoite surface antigen (MSA-2) of the malaria parasite Plasmodium falciparum

1991 ◽  
Vol 11 (2) ◽  
pp. 963-971
Author(s):  
B Fenton ◽  
J T Clark ◽  
C M Khan ◽  
J V Robinson ◽  
D Walliker ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Surface Antigen ◽  
Dna Sequences ◽  
Amino Acid Sequences ◽  
Merozoite Surface Antigen ◽  
Parasite Plasmodium ◽  
Genes Encoding ◽  
Parasite Plasmodium Falciparum ◽  
Group B

Merozoite surface antigen MSA-2 of the human parasite Plasmodium falciparum is being considered for the development of a malaria vaccine. The antigen is polymorphic, and specific monoclonal antibodies differentiate five serological variants of MSA-2 among 25 parasite isolates. The variants are grouped into two major serogroups, A and B. Genes encoding two different variants from serogroup A have been sequenced, and their DNA together with deduced amino acid sequences were compared with sequences encoded by other alleles. The comparison shows that the serological classification reflects differences in DNA sequences and deduced primary structure of MSA-2 variants and serogroups. Thus, the overall homologies of DNA and amino acid sequences are over 95% among variants in the same serogroup. In contrast, similarities between the group A variants and a group B variant are only 70 and 64% for DNA and amino acid sequences, respectively. We propose that the MSA-2 protein is encoded by two highly divergent groups of alleles, with limited additional polymorphism displayed within each group.

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