Rubén Salcedo-Hernández

Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis

ABSTRACT The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5αF′. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2°C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.

Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis

Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.

Enhanced synthesis and antimicrobial activities of bacteriocins produced by Mexican strains of Bacillus thuringiensis

Recently, we reported the synthesis of five bacteriocin-like inhibitor substances (Bt-BLIS: morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) by Mexican strains of Bacillus thuringiensis. Here we show that, collectively, these Bt-BLIS have a moderate to broad spectrum of antibacterial activity, being toxic to clinically significant against Gram-positive and Gram-negative bacteria, including common etiological agents of human diseases, such as strep throat and scarlet fever, septicemia, pneumonia, urinary tract infection, and emetic and gastrointestinal syndromes. Although synthesis of the five Bt-BLIS was independent of the presence of a target inducing bacterium, we demonstrated for the first time that a proteinaceous component(s) secreted by, or liberated by proteolytic cleavage of Bacillus cereus 183 following treatment with proteinase K, enhanced Bt-BLIS synthesis.

Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74.

Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine–Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.

Molecular Cloning and Purification of an Endochitinase From Serratia marcescens (Nima)

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5αF′, and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5′-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0–10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C.

Isolation of a new Mexican strain of Bacillus subtilis with antifungal and antibacterial activities.

Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants.

Generation of chitin-derived oligosaccharides toxic to pathogenic bacteria using ChiA74, an endochitinase native to Bacillus thuringiensis

Aims:  To demonstrate that an endochitinase (ChiA74) native to Bacillus thuringiensis can be used to generate chitin‐derived oligosaccharides (OGS) with antibacterial activity against a number of aetiological agents of disease, including bacteria that cause diarrhoeal and emetic syndromes in humans.

Possible role of ionic gradients in the apical growth of Neurospora crassa

The effects of the Ca2+/H+ exchanger A23187 and the K+/H+ exchanger nigericin on the growth of Neurospora crassa were analyzed. Both ionophores had the same effects on the fungus. They both inhibited growth in liquid media, apical extension being more affected than protein synthesis. A sudden challenge to either ionophore on solid media rapidly stopped hyphal extension. Additionally, both ionophores induced profuse mycelium branching and upward hyphal growth. Hyphae growing on nigericin-containing media also burst at the apex. Both ionophores caused a rapid inhibition in the apically-occurring synthesis of structural wall polysaccharides, but they did not affect mitochondrial energy conservation. With the use of DiBAC, a membrane-potential sensitive fluorophore, it was excluded that their effects were due to depletion of the plasma membrane potential. Considering that both ionophores exchange H+ for different metallic ions, we concluded that their effect was due to dissipation of a proton gradient, which is directly or indirectly involved in the apical growth of the fungus.

Bacillus thuringiensis subsp. israelensis producing endochitinase ChiA74Δsp inclusions and its improved activity against Aedes aegypti.

The objective of this study was to produce stable inclusions of chitinase ChiA74Δsp in Bacillus thuringiensis subsp. israelensis (Bti) and to assay its insecticidal activity against Aedes aegypti larvae.

Organization and regulation of the mitochondrial oxidative pathway in Mucor rouxii

Summary: Mitochondria from spores, anaerobic yeasts and aerobic mycelium of Mucor rouxii were isolated and the components of the respiratory pathways were analysed. Our data suggest the presence in these mitochondria of three different NADH dehydrogenases, three b- and two c-type cytochromes, plus three different terminal oxidases: cytochrome c oxidase, an incompletely characterized cytochrome which, by its spectral characteristics, behaves as a cytochrome o, and a salicylhydroxamic-acid-sensitive oxidase. The composition of the respiratory chain, as well as the relative proportions of cytochromes and oxidases in the mitochondria, were distinct at each developmental stage. Our data suggest that the development of the respiratory pathways during differentiation is mainly regulated by the oxygen tension. Besides this role, oxygen also influences the relative number of mitochondria in the cells and their ultrastructural organization.