Small, hydrophobic proteins whose synthesis is repressed by small RNAs (sRNAs), denoted type I toxin–antitoxin modules, were first discovered on plasmids where they regulate plasmid stability, but were subsequently found on a few bacterial chromosomes. We used exhaustive PSI-BLAST and TBLASTN searches across 774 bacterial genomes to identify homologs of known type I toxins. These searches substantially expanded the collection of predicted type I toxins, revealed homology of the Ldr and Fst toxins, and suggested that type I toxin–antitoxin loci are not spread by horizontal gene transfer. To discover novel type I toxin–antitoxin systems, we developed a set of search parameters based on characteristics of known loci including the presence of tandem repeats and clusters of charged and bulky amino acids at the C-termini of short proteins containing predicted transmembrane regions. We detected sRNAs for three predicted toxins from enterohemorrhagic Escherichia coli and Bacillus subtilis, and showed that two of the respective proteins indeed are toxic when overexpressed. We also demonstrated that the local free-energy minima of RNA folding can be used to detect the positions of the sRNA genes. Our results suggest that type I toxin–antitoxin modules are much more widely distributed among bacteria than previously appreciated.

SUMMARY: There has been a great expansion in the number of small regulatory RNAs identified in bacteria. Some of these small RNAs repress the synthesis of potentially toxic proteins. Generally the toxin proteins are hydrophobic and less than 60 amino acids in length, and the corresponding antitoxin small RNA genes are antisense to the toxin genes or share long stretches of complementarity with the target mRNAs. Given their short length, only a limited number of these type I toxin-antitoxin loci have been identified, but it is predicted that many remain to be found. Already their characterization has given insights into regulation by small RNAs, has suggested functions for the small toxic proteins at the cell membrane, and has led to practical applications for some of the type I toxin-antitoxin loci.

Acid adaptation ofStreptococcus mutansUA159 involves several different mechanisms, including the ability to alter its proportion of long-chain, monounsaturated membrane fatty acids (R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). In the present study, we examined the mechanism and timing of changes in fatty acid ratios and the potential benefit that an increased proportion of long-chained fatty acids has for the organism during growth at low pH. Cells taken from steady-state cultures at intermediate pH values of 6.5, 6, and 5.5 showed incremental changes from the short-chained, saturated membrane fatty acid profile normally seen in pH 7 cultures to the long-chained, monounsaturated fatty acids more typically observed in acidic cultures (pH 5). Our observations showed that the bacterium was capable of effecting the majority of changes in approximately 20 min, far less than one generation time. However, reversion to the distribution of fatty acids seen in cells growing at a pH of 7 required a minimum of 10 generations. Fatty acid composition analysis of cells taken from cultures treated with chloramphenicol suggested that the changes in fatty acid distribution did not require de novo protein synthesis. Cells treated with the fatty acid biosynthesis inhibitor cerulenin were unable to alter their membrane fatty acid profiles and were unable to survive severe acidification. Results presented here indicate that membrane fatty acid redistribution is important for low pH survival and, as such, is a component of theS. mutansacid-adaptation arsenal.

Summary: The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, although the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five‐sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18‐ to 19‐amino‐acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL‐acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell.

Previously, it has been demonstrated that the membrane fatty acid composition of Streptococcus mutans is affected by growth pH (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70: 929-936, 2004; R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189: 89-92, 2000). Specifically, the proportion of monounsaturated fatty acids increases when the organism is grown in acidic environments; if the shift to increased monounsaturated fatty acids is blocked by the addition of a fatty acid biosynthesis inhibitor, the organism is rendered more acid sensitive (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70: 929-936, 2004). Recently, work with Streptococcus pneumoniae has identified a novel enzyme, FabM, responsible for the production of monounsaturated fatty acids (H. Marrakchi, K. H. Choi, and C. O. Rock, J. Biol. Chem. 277: 44809-44816, 2002). Using the published S. pneumoniae sequence, a putative FabM was identified in the S. mutans strain UA159. We generated a fabM strain that does not produce unsaturated fatty acids as determined by gas chromatography of fatty acid methyl esters. The mutant strain was extremely sensitive to low pH in comparison to the wild type; however, the acid-sensitive phenotype was relieved by growth in the presence of long-chain monounsaturated fatty acids or through genetic complementation. The strain exhibited reduced glycolytic capability and altered glucose-PTS activity. In addition, the altered membrane composition was more impermeable to protons and did not maintain a normal ΔpH. The results suggest that altered membrane composition can significantly affect the acid survival capabilities, as well as several enzymatic activities, of S. mutans .