ABSTRACT
Four closely related peptides were isolated from seed of Impatiens balsamina and were shown to be inhibitory to the growth of a range of fungi and bacteria, while not being cytotoxic to cultured human cells. The peptides, designated Ib-AMP1, Ib-AMP2, Ib-AMP3, and Ib-AMP4, are 20 amino acids long and are the smallest plant-derived antimicrobial peptides isolated to date. The Ib-AMPs (I. balsamina antimicrobial peptides) are highly basic and contain four cysteine residues which form two intramolecular disulfide bonds. Searches of protein data bases have failed to identify any proteins with significant homology to the peptides described here. Characterization of isolated cDNAs reveals that all four peptides are encoded within a single transcript. The predicted Ib-AMP precursor protein consists of a prepeptide followed by 6 mature peptide domains, each flanked by propeptide domains ranging from 16 to 35 amino acids in length. Such a primary structure with repeated alternating basic mature peptide domains and acidic propeptide domains has, to date, not been reported in plants.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Cysteine/analysis , Plant Proteins/isolation & purification , Plants/chemistry , Seeds/chemistry , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Base Sequence , Cells, Cultured , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Erythrocytes/drug effects , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Humans , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/pharmacology , Sequence AlignmentABSTRACT
Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination.
Subject(s)
Bacillus subtilis/classification , Genes, Bacterial , Rec A Recombinases/genetics , Recombination, Genetic , Alleles , Bacillus subtilis/genetics , Colony Count, Microbial , Mutation , Transformation, GeneticABSTRACT
We determined the effect of various Bacillus subtilis dna(Ts) mutations on pUB110 and chromosomal replication. Leading strand DNA synthesis of pUB110, starting by a nick at the plasmid replication origin (oriU), is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in thermosensitive mutants dnaD, dnaF, dnaH and dnaN known to cause thermosensitivity of the various subunits of DNA polymerase III. When the lagging strand origin (oriL) is exposed, the DnaG protein (DNA primase) alone, or in association with unknown protein(s) binds asymmetrically to oriL to form a primer that is also extended by DNA polymerase III. In oriL- plasmids like pBT32, leading and lagging strand DNA syntheses are decoupled from each other. The DnaB protein, that is not required for pUB110 replication, may be associated with priming at a second unidentified lagging strand origin on pBT32. At non-permissive temperature, the dnaC30 and dnaI2 mutations affect both pUB110 and chromosomal DNA synthesis.
Subject(s)
Bacillus subtilis/genetics , DNA Replication , DNA, Bacterial/biosynthesis , Mutation , Plasmids , Temperature , Chloramphenicol/pharmacology , DNA Polymerase III/metabolism , DNA, Bacterial/physiology , Genes, Bacterial , Models, Genetic , Protein Synthesis Inhibitors/pharmacology , Rifampin/pharmacology , Transformation, GeneticABSTRACT
An isogenic set of "prophage-free," DNA repair-proficient and -deficient strains of Bacillus subtilis were characterized phenotypically. The mutant strains were provisionally classified into four categories on the basis of their sensitivity to DNA-damaging agents, their ability to release phage after lysogenization followed by damage to chromosomal DNA, and their impairment in genetic exchange. The properties of double Rec- mutants showed that recF and addA belong to different epistatic groups, whereas recF, recL, and recH fall into the same group. More than one pathway for genetic exchange might be operative in B. subtilis.
Subject(s)
Bacillus subtilis/genetics , DNA Damage , DNA Repair , DNA, Bacterial/drug effects , Recombination, Genetic , Bacterial Proteins/genetics , Ethyl Methanesulfonate/pharmacology , Methyl Methanesulfonate/pharmacology , Mutation , SOS Response, Genetics , Transfection , Transformation, Bacterial , Virus ActivationABSTRACT
By deletion analysis we have defined a 1.1 kb segment required for driving autonomous replication of the plasmid pC194. The minimal replicon specifies a positive, RepH, and a negative, Inc8A, trans-acting product and their target sites. The RepH product has a Mr of 34.1 kDa, could be overproduced, and binds specifically to the pC194 origin region. By trans complementation studies we have shown that pC194 replication is indirectly controlled at the level of RepH synthesis by a negative product, IncA, that is transcribed within the repH transcription unit in the opposite direction ("antisense RNA"). The antisense RNA regulates the RepH synthesis by a mechanism that seems to involve RNA/RNA interaction in a manner that interferes with translation. In addition, an autoregulatory control might be operative.
Subject(s)
Bacillus subtilis/genetics , DNA Replication , DNA, Bacterial/genetics , Plasmids , Regulatory Sequences, Nucleic Acid , Bacterial Proteins/physiology , DNA-Binding Proteins/physiology , Gene Expression Regulation , Genes, BacterialABSTRACT
An isogenic set of 11 recombination-deficient mutant strains of Bacillus subtilis has been constructed. Whereas plasmid pUB110 is stably maintained in such Rec- cells, the high copy number plasmid pC194 is unstable. Instability in Rec- strains could be mostly attributed to the deleterious effect of the presence of the plasmid on the Rec- cells' growth capability. In part, instability of pC194 derivatives could also be correlated with the presence of an unusually high amount of multimeric DNA molecules.
