ABSTRACT
In seawater, enteric bacteria evolve toward a stressed state that is difficult to identify because of major alterations of their phenotype. In this study, we incubated four reference strains of S. enterica serovar Typhimurium in seawater microcosms for 10 months and studied the modifications of their main phenotypic characters. All of the strains lost some key characters used for traditional identification of the Salmonella genus. They became able to produce acetoin, and tryptophane deaminase activity became positive, but they lost the capacity to use rhamnose. We were able to show some modifications of the level of enzymatic profile as well as in their antibiotic susceptibility. The atypical cells of S. enterica serovar Typhimurium were identified by polymerase chain reaction (PCR) methods using the internal transcribed spacer region, and they were confirmed by multiplex PCR after the simultaneous amplification of the phoP, Hin, and H-li genes.
Subject(s)
Bacterial Typing Techniques , Heat-Shock Response , Salmonella typhimurium/classification , Salmonella typhimurium/physiology , Seawater/microbiology , Adaptation, Physiological , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Ecosystem , Humans , Microbial Sensitivity Tests , Phenotype , Polymerase Chain Reaction/methods , Salmonella typhimurium/drug effects , Salmonella typhimurium/geneticsABSTRACT
A barley cDNA clone encoding a cysteine proteinase inhibitor was characterized. The deduced amino acid sequence of this barley cystatin (Hv-CPI) contains the motif QXVXG conserved among members of the cystatin superfamily. The gene (Icy), located on chromosome 2, was expressed in embryos, developing endosperms, leaves and roots as assessed by northern blot analysis. Western blot analysis detected a slightly retarded band in leaves that was not present in roots or seeds. In these two organs a more precise location of Hv-CPI was done by immuno-histochemical analysis, with polyclonal antibodies raised against the recombinant CPI protein expressed in Escherichia coli. This protein efficiently inhibited papain (Ki 2.0 x 10(-8) M) and ficin (Ki 2.2 x 10(-8) M) and, to a lesser extent, chymopapain (Ki 1.6 x 10(-7) M) and was inactive against bromelain. The Icy mRNA expression in vegetative tissues increased in response to anaerobiosis, dark and cold shock (6 degrees C).
Subject(s)
Cystatins/genetics , Cysteine Proteinase Inhibitors/genetics , Hordeum/genetics , Plant Proteins/genetics , Amino Acid Motifs , Amino Acid Sequence , Anaerobiosis , Base Sequence , Blotting, Northern , Blotting, Southern , Blotting, Western , Cold Temperature , Conserved Sequence , Cystatins/metabolism , Cysteine Proteinase Inhibitors/metabolism , DNA, Complementary/analysis , DNA, Complementary/isolation & purification , Darkness , Hordeum/metabolism , Hordeum/physiology , Immunohistochemistry , Molecular Sequence Data , Papain/antagonists & inhibitors , Plant Proteins/isolation & purification , Plant Proteins/metabolismABSTRACT
We have isolated several herbicide-resistant cell lines from photosynthetic cell suspensions of soybean (Glycine max) that possessed different levels of herbicide resistance, photosystem II activity, and chlorophyll a/b ratio. We have further studied the STR7 mutant, which showed the highest level of resistance to atrazine as well as a cross-resistance to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (50- and 3-fold, respectively, compared with the wild type). Sequencing of the psbA gene (coding for the D1 polypeptide of photosystem II) from this mutant revealed a single change, serine-268 to proline, in the D1 protein. To our knowledge, this substitution has not previously been described in any photosynthetic organism. In addition to affecting atrazine resistance, this single amino acid change caused a decrease in the electron transfer rate between the secondary acceptors QA and QB and a stabilization of the S2QB- and S3QB- states. The mutant also showed a larger antenna size, an increase in non-QB-reducing centers, and a higher sensitivity to light stress. The unusual stability of the S2QB- and S3QB- states indicates that STR7 belongs to a new class of QB-site mutants.
