John R. Roth

Gut inflammation provides a respiratory electron acceptor for Salmonella

Salmonella enterica serotype Typhimurium (S. Typhimurium) causes acute gut inflammation by using its virulence factors to invade the intestinal epithelium and survive in mucosal macrophages. The inflammatory response enhances the transmission success of S. Typhimurium by promoting its outgrowth in the gut lumen through unknown mechanisms. Here we show that reactive oxygen species generated during inflammation react with endogenous, luminal sulphur compounds (thiosulphate) to form a new respiratory electron acceptor, tetrathionate. The genes conferring the ability to use tetrathionate as an electron acceptor produce a growth advantage for S. Typhimurium over the competing microbiota in the lumen of the inflamed gut. We conclude that S. Typhimurium virulence factors induce host-driven production of a new electron acceptor that allows the pathogen to use respiration to compete with fermenting gut microbes. Thus the ability to trigger intestinal inflammation is crucial for the biology of this diarrhoeal pathogen.

Genetic engineering in vivo using translocatable drug-resistance elements. New methods in bacterial genetics.

Abstract A number of translocatable drug-resistance elements have recently been described which are able to insert themselves into a large number of different sites in prokaryotic genomes. These elements cause recognizable mutations when insertion occurs within a structural gene or an operon. Drug-resistance elements are also associated with other kinds of illegitimate recombination events, notably deletions and inversions. This paper summarizes uses to which these properties of translocatable drugr-esistance elements can be put in genetic manipulations of bacteria. Translocatable drug-resistance elements are useful in isolation of mutants (even where the mutant phenotype is not easily scored), in the construction of strains and other genetic manipulations (even when selection is difficult or impossible), in localized mutagenesis, in chromosomal mapping, in construction of Hfr strains with known origin and direction of chromosome transfer, in complementation tests, in construction of new F′ plasmids, in construction of new specialized transducing phages, in isolation of deletions with one or both endpoints specified, in construction of gene and operon fusions, and in the selection and maintenance of chromosomal duplications. Experiments are described which illustrate many of these techniques.

Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota

Conventional wisdom holds that microbes support their growth in vertebrate hosts by exploiting a large variety of nutrients. We show here that use of a specific nutrient (ethanolamine) confers a marked growth advantage on Salmonella enterica serovar Typhimurium (S. Typhimurium) in the lumen of the inflamed intestine. In the anaerobic environment of the gut, ethanolamine supports little or no growth by fermentation. However, S. Typhimurium is able to use this carbon source by inducing the gut to produce a respiratory electron acceptor (tetrathionate), which supports anaerobic growth on ethanolamine. The gut normally converts ambient hydrogen sulfide to thiosulfate, which it then oxidizes further to tetrathionate during inflammation. Evidence is provided that S. Typhimuriums growth advantage in an inflamed gut is because of its ability to respire ethanolamine, which is released from host tissue, but is not utilizable by competing bacteria. By inducing intestinal inflammation, S. Typhimurium sidesteps nutritional competition and gains the ability to use an abundant simple substrate, ethanolamine, which is provided by the host.

Ohno's dilemma: Evolution of new genes under continuous selection

New genes with novel functions arise by duplication and divergence, but the process poses a problem. After duplication, an extra gene copy must rise to sufficiently high frequency in the population and remain free of common inactivating lesions long enough to acquire the rare mutations that provide a new selectable function. Maintaining a duplicated gene by selection for the original function would restrict the freedom to diverge. (We refer to this problem as Ohnos dilemma). A model is described by which selection continuously favors both maintenance of the duplicate copy and divergence of that copy from the parent gene. Before duplication, the original gene has a trace side activity (the innovation) in addition to its original function. When an altered ecological niche makes the minor innovation valuable, selection favors increases in its level (the amplification), which is most frequently conferred by increased dosage of the parent gene. Selection for the amplified minor function maintains the extra copies and raises the frequency of the amplification in the population. The same selection favors mutational improvement of any of the extra copies, which are not constrained to maintain their original function (the divergence). The rate of mutations (per genome) that improve the new function is increased by the multiplicity of target copies within a genome. Improvement of some copies relaxes selection on others and allows their loss by mutation (becoming pseudogenes). Ultimately one of the extra copies is able to provide all of the new activity.

Amplification-mutagenesis: Evidence that ''directed'' adaptive mutation and general hypermutability result from growth with a selected gene amplification

When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation). This observation suggested that growth limitation stimulates mutability. Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps. First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac+ revertant allele within the amplified array. Finally, it favors loss of mutant copies until a stable haploid lac+ revertant arises and overgrows the colony. By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone. This sequence of events appears to direct mutations to useful sites. General mutagenesis is a side-effect of growth with an amplification (SOS induction). The F′ plasmid, which carries lac, contributes by stimulating gene duplication and amplification. Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose. The sequence of events—amplification, mutation, segregation—may help to explain both the origins of some cancers and the evolution of new genes under selection.

Histidine Regulatory Mutants in Salmonella typhimurium I. Isolation and General Properties

At least four genetically distinct classes of mutations give rise to triazolealanine resistance in Salmonella typhimurium. Each class studied affects regulation of the histidine operon. Mutants of one class, hisO, are located at one end of the histidine operon. This class may be similar to operator constitutive mutants studied in other systems. At least two genetic sites are present in this region. A second class, hisS, is linked in transduction tests to the guaA and strB loci. This class contains a low specific activity of histidyl-tRNA synthetase and may represent mutations in the structural gene for this enzyme Roth & Ames 1966. Mutations of a third class, hisR, are linked in transduction tests to the ilv and metE loci Roth & Hartman 1965 and affect the levels of effective transfer RNA his Silbert, Fink & Ames 1966. A fourth class of mutants, hisT, is linked to the purF and aroD loci.

Genomic buffering mitigates the effects of deleterious mutations in bacteria

The relationship between the number of randomly accumulated mutations in a genome and fitness is a key parameter in evolutionary biology. Mutations may interact such that their combined effect on fitness is additive (no epistasis), reinforced (synergistic epistasis) or mitigated (antagonistic epistasis). We measured the decrease in fitness caused by increasing mutation number in the bacterium Salmonella typhimurium using a regulated, error-prone DNA polymerase (polymerase IV, DinB). As mutations accumulated, fitness costs increased at a diminishing rate. This suggests that random mutations interact such that their combined effect on fitness is mitigated and that the genome is buffered against the fitness reduction caused by accumulated mutations. Levels of the heat shock chaperones DnaK and GroEL increased in lineages that had accumulated many mutations, and experimental overproduction of GroEL further increased the fitness of lineages containing deleterious mutations. These findings suggest that overexpression of chaperones contributes to antagonistic epistasis.

Conserving a Volatile Metabolite: a Role for Carboxysome-Like Organelles in Salmonella enterica

Salmonellae can use ethanolamine (EA) as a sole source of carbon and nitrogen. This ability is encoded by an operon (eut) containing 17 genes, only 6 of which are required under standard conditions (37 degrees C; pH 7.0). Five of the extra genes (eutM, -N, -L, -K, and -G) become necessary under conditions that favor loss of the volatile intermediate, acetaldehyde, which escapes as a gas during growth on EA and is lost at a higher rate from these mutants. The eutM, -N, -L, and -K genes encode homologues of shell proteins of the carboxysome, an organelle shown (in other organisms) to concentrate CO(2). We propose that carboxysome-like organelles help bacteria conserve certain volatile metabolites-CO(2) or acetaldehyde-perhaps by providing a low-pH compartment. The EutG enzyme converts acetaldehyde to ethanol, which may improve carbon retention by forming acetals; alternatively, EutG may recycle NADH within the carboxysome.

The Alternative Electron Acceptor Tetrathionate Supports B12-Dependent Anaerobic Growth of Salmonella enterica Serovar Typhimurium on Ethanolamine or 1,2-Propanediol

Synthesis of cobalamin de novo by Salmonella enterica serovar Typhimurium strain LT2 and the absence of this ability in Escherichia coli present several problems. This large synthetic pathway is shared by virtually all salmonellae and must be maintained by selection, yet no conditions are known under which growth depends on endogenous B12. The cofactor is required for degradation of 1,2-propanediol and ethanolamine. However, cofactor synthesis occurs only anaerobically, and neither of these carbon sources supports anaerobic growth with any of the alternative electron acceptors tested thus far. This paradox is resolved by the electron acceptor tetrathionate, which allows Salmonella to grow anaerobically on ethanolamine or 1,2-propanediol by using endogenously synthesized B12. Tetrathionate provides the only known conditions under which simple cob mutants (unable to make B12) show a growth defect. Genes involved in this metabolism include the ttr operon, which encodes tetrathionate reductase. This operon is globally regulated by OxrA (Fnr) and induced anaerobically by a two-component system in response to tetrathionate. Salmonella reduces tetrathionate to thiosulfate, which it can further reduce to H2S, by using enzymes encoded by the genes phs and asr. The genes for 1,2-propanediol degradation (pdu) and B12 synthesis (cob), along with the genes for sulfur reduction (ttr, phs, and asr), constitute more than 1% of the Salmonella genome and are all absent from E. coli. In diverging from E. coli, Salmonella acquired some of these genes unilaterally and maintained others that are ancestral but have been lost from the E. coli lineage.

Mechanisms of Suppression

Publisher Summary This chapter discusses that the suppressor mutations are one class of secondary mutations that modify the phenotype in the presence of the originally mutant gene. In contrast to “enhancers” that make the mutant phenotype more extreme, suppressor mutations yield organisms phenotypically more like the wild type: The mutant phenotype is “suppressed.” Thus, suppressors are mutations that elicit a revertant or partially revertant phenotype. But suppressor mutations can be genetically separated, by recombination, from the mutations that they suppress. This review is intended as a supplement to some condensed reviews and more recent extensive summaries restricted to aspects of informational suppression. The purpose of this chapter is to describe systems that seem to be informative and/or illustrative of types, of suppressor activity. It also highlights that suppressor analysis can yield insight into arrays of problems, not readily subject to more classic genetic experimentation. Contributions to genetics and the resolving power of suppressor studies also are discussed in this chapter.