Hinnavin I, an antibacterial peptide from cabbage butterfly, Artogeia rapae.

We have previously isolated four antibacterial peptides from the immune haemolymph of the fifth instar larvae of cabbage butterfly, Artogeia rapae [Yoe, S. M., Bang, I. S., Kang, C. S., and Kim, H. J. (1996) Mol. Cells 6, 609-614]. They were induced by live, nonpathogenic gram negative bacteria. One of these novel antibacterial peptides was named hinnavin I. Hinnavin I is heat stable; its activity was retained after 60 min incubation at 100 degrees C, being effective against gram negative and/or gram positive bacteria. Hinnavin I and lysozyme II showed a powerful synergistic effect on the inhibition of bacterial growth. Amino acid composition was analyzed and the molecular weight was determined to be 4,139.94+/-10.91 Da by the ESI mass spectrometer. To elucidate the primary structure of hinnavin I, the amino acid sequence of this peptide was determined by N-terminal sequencing techniques. The amino-terminal half of the molecule was rich in charged amino acids and was hydrophilic, whereas the carboxyl-terminal half was hydrophobic.

Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium.

Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and sigma s-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli. The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.

OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium.

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.

Regulatory circuits involved with pH-regulated gene expression in Salmonella typhimurium.

Salmonella typhimurium encounters a variety of acid conditions during both its natural and pathogenic existence. The ability of this organism to respond transcriptionally to low pH is an area of active interest but little knowledge. As part of an ongoing investigation of low-pH adaptation, 18 pH-controlled lacZ operon fusions in Salmonella typhimurium have been identified (15 in this study) and categorized into at least 11 different loci. They include iroA (at 57 min), aciA (99 min), aciB (90-93 min), aciD (ompC, 45 min), aciJ, aciK (33-36 min), aniC (93 min), anil (33-36 min), hyd (59 min), cadA (54 min) and aniG (63 min). All but two were induced by low pH. One of the exceptions, the iron-regulated iroA locus, was induced at high pH. The unusual aciA locus was induced by low pH under semiaerobic conditions but high pH under aerobic conditions. Most of the other aci genes were expressed best under anaerobic conditions. Many of these genes exhibited strict co-inducer requirements for small molecules to be expressed in minimal medium. These included iron for iroA, tyrosine for aniC, I and aciK, mannose for aniG, formate for hyd, lysine for cadA, and unknown components of complex medium for aciA, aciB and aciD. Six regulatory circuits were revealed involving at least five regulatory loci (fur, oxrG, earAB, earC and ompR). As part of the adaptive response to low pH, S. typhimurium will induce an acid protection system called the acid tolerance response (ATR). As has been shown for fur mutations, the oxrG regulatory mutation interfered with the normal induction of this system.

cDNA cloning and antibacterial activities of cecropin D-like peptides from Agrius convolvuli.

We have characterized full-length cDNAs encoding two isoforms of agriusin, cecropin D-like antibacterial peptide, present in the hemolymph of the immunized Agrius convolvuli larvae. The cloned cDNAs of agriusins 1 and 2 contain 331 and 329 bp, respectively. The nucleotide sequencing of cDNAs showed that they encode 62 amino acids, whose mature portion was deduced to consist of 38 amino acid residues with over 94% sequence identity. In the sequence homology search, mature agriusin 1 showed over 86 and 71% amino acid sequence homology with bactericidin 4 from Manduca sexta and cecropin D from Hyalophora cecropia, respectively. Since it was demonstrated from the deduced amino acid sequences that the C-terminal residues of agriusins are followed by a Gly residue, two types of synthetic agriusin 1 (syn-agriusin 1 amide and acid) were prepared to verify if natural agriusin 1 is C-terminally amidated. From acid-urea PAGE and reversed phase HPLC profiles to compare two synthetic peptides, we could confirm that the C-terminal amino acid residue of natural agriusin 1, like several cecropins so far identified, is amidated. Finally, our antibacterial assay performed with two syn-agriusins 1 revealed that there is little difference between antibacterial activities of both peptides against Gram-positive and Gram-negative bacteria.

Expression of cspH, encoding the cold shock protein in Salmonella enterica serovar Typhimurium UK-1.

Both Salmonella enterica serovar Typhimurium and Escherichia coli contain the cspH gene encoding CspH, one of the cold shock proteins (CSPs). In this study, we investigated the expression of cspH in S. enterica serovar Typhimurium and found that it was induced in response to a temperature downshift during exponential phase. The cspH promoter was activated at 37 degrees C, and its mRNA was more stable than the other csp mRNAs at 37 degrees C. Moreover, lacZ expression of the translational cspH-lacZ fusion was induced at that temperature. Interestingly, the cspH mRNA had a much shorter 5'-untranslated region than those in the other cold-shock-inducible genes, and the promoter sequence, which was only 55 bp, was sufficient for cspH expression. The 14-base downstream box located 12 bases downstream of the initiation codon of cspH mRNA was essential for its cold shock activation.