Jitesh A. Soares

SdiA, an N-acylhomoserine lactone receptor, becomes active during the transit of Salmonella enterica through the gastrointestinal tract of turtles

Background LuxR-type transcription factors are typically used by bacteria to determine the population density of their own species by detecting N-acylhomoserine lactones (AHLs). However, while Escherichia and Salmonella encode a LuxR-type AHL receptor, SdiA, they cannot synthesize AHLs. In vitro, it is known that SdiA can detect AHLs produced by other bacterial species. Methodology/Principal Findings In this report, we tested the hypothesis that SdiA detects the AHL-production of other bacterial species within the animal host. SdiA did not detect AHLs during the transit of Salmonella through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during the transit of Salmonella through turtles. All turtles examined were colonized by the AHL-producing species Aeromonas hydrophila. Conclusions/Significance We conclude that the normal gastrointestinal microbiota of most animal species do not produce AHLs of the correct type, in an appropriate location, or in sufficient quantities to activate SdiA. However, the results obtained with turtles represent the first demonstration of SdiA activity in animals.

Salmonella enterica serovar Typhimurium can detect acyl homoserine lactone production by Yersinia enterocolitica in mice.

LuxR-type transcription factors detect acyl homoserine lactones (AHLs) and are typically used by bacteria to determine the population density of their own species. Escherichia coli and Salmonella enterica serovar Typhimurium cannot synthesize AHLs but can detect the AHLs produced by other bacterial species using the LuxR homolog, SdiA. Previously we determined that S. Typhimurium did not detect AHLs during transit through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during transit through turtles colonized by Aeromonas hydrophila, leading to the hypothesis that SdiA is used for detecting the AHL production of other pathogens. In this report, we determined that SdiA is activated during the transit of S. Typhimurium through mice infected with the AHL-producing pathogen Yersinia enterocolitica. SdiA is not activated during transit through mice infected with a yenI mutant of Y. enterocolitica that cannot synthesize AHLs. However, activation of SdiA did not confer a fitness advantage in Yersinia-infected mice. We hypothesized that this is due to infrequent or short interactions between S. Typhimurium and Y. enterocolitica or that the SdiA regulon members do not function in mice. To test these hypotheses, we constructed an S. Typhimurium strain that synthesizes AHLs to mimic a constant interaction with Y. enterocolitica. In this background, sdiA(+) S. Typhimurium rapidly outcompetes the sdiA mutant in mice. All known members of the sdiA regulon are required for this phenotype. Thus, all members of the sdiA regulon are functional in mice.

E. coli K-12 and EHEC Genes Regulated by SdiA

Background Escherichia and Salmonella encode SdiA, a transcription factor of the LuxR family that regulates genes in response to N-acyl homoserine lactones (AHLs) produced by other species of bacteria. E. coli genes that change expression in the presence of plasmid-encoded sdiA have been identified by several labs. However, many of these genes were identified by overexpressing sdiA on a plasmid and have not been tested for a response to sdiA produced from its natural position in the chromosome or for a response to AHL. Methodology/Principal Findings We determined that two important loci reported to respond to plasmid-based sdiA, ftsQAZ and acrAB, do not respond to sdiA expressed from its natural position in the chromosome or to AHLs. To identify genes that are regulated by chromosomal sdiA and/or AHLs, we screened 10,000 random transposon-based luciferase fusions in E. coli K-12 and a further 10,000 in E. coli O157:H7 for a response to AHL and then tested these genes for sdiA-dependence. We found that genes encoding the glutamate-dependent acid resistance system are up-regulated, and fliE is down-regulated, by sdiA. Gene regulation by sdiA of E. coli is only partially dependent upon AHL. Conclusions/Significance The genes of E. coli that respond to plasmid-based expression of sdiA are largely different than those that respond to chromosomal sdiA and/or AHL. This has significant implications for determining the true function of AHL detection by E. coli.

The Residue Mass of L-Pyrrolysine in Three Distinct Methylamine Methyltransferases

Single in-frame amber (UAG) codons are found in the genes encoding MtmB, MtbB, or MttB, the methyltransferases initiating methane formation from monomethylamine, dimethylamine, or trimethylamine, respectively, in certain Archaea. The crystal structure of MtmB demonstrated that the amber codon codes for pyrrolysine, the 22nd genetically encoded amino acid found in nature. Previous attempts to visualize the amber-encoded residue by mass spectrometry identified only lysine, leaving information on the existence and structure of pyrrolysine resting entirely on crystallography of a single protein. Here we report successful mass spectral characterization of naturally occurring pyrrolysine and the first demonstration of the amber-encoded residue in proteins other than MtmB. The sequencing of chymotryptic fragments from acetonitrile-denatured proteins by tandem mass spectrometry revealed the mass of the amber-encoded residue in MtmB, MtbB, and MttB as 237.2 ± 0.2 Da. Fourier transform ion cyclotron resonance mass spectrometry produced an accurate measurement for the pyrrolysyl-residue as 237.1456 Da, within error limits of the predicted mass based on the empirical formula C12H19N3O2. These measurements support the structure of pyrrolysine in MtmB as 4-methylpyrroline-5-carboxylate in amide linkage with the ϵN of lysine but not the alternative structure in which the 4-substituent of the pyrroline ring is an amine group. The presence of pyrrolysine with statistically identical mass in all three methyltransferases is in keeping with the proposed direct incorporation of pyrrolysine into protein during translation of the UAG codon and suggests that MtbB and MttB may exploit the unusual electrophilicity of pyrrolysine during catalysis.

Characterization of a Methanosarcina acetivorans mutant unable to translate UAG as pyrrolysine

The methyltransferases initiating methanogenesis from trimethylamine, dimethylamine and monomethylamine possess a novel residue, pyrrolysine. Pyrrolysine is the 22nd amino acid, because it is encoded by a single amber (UAG) codon in methylamine methyltransferase transcripts. A dedicated tRNACUA for pyrrolysine, tRNAPyl, is charged by a pyrrolysyl‐tRNA synthetase with pyrrolysine. As the first step towards the genetic analysis of UAG translation as pyrrolysine, a 761 base‐pair genomic segment in Methanosarcina acetivorans containing the pylT gene (encoding tRNAPyl) was deleted and replaced by a puromycin resistance cassette. The ΔppylT mutant lacks detectable tRNAPyl, but grows as wild‐type on methanol or acetate. Unlike wild‐type, the ΔppylT strain cannot grow on any methylamine, nor use monomethylamine as sole nitrogen source. Wild‐type cells, but not ΔppylT, have monomethylamine methyltransferase activity during growth on methanol. Immunoblot analysis indicated monomethylamine methyltransferase was absent in ΔppylT. The phenotype of ΔppylT reveals the deficiency in methylamine metabolism expected of a Methanosarcina species unable to decode UAG codons as pyrrolysine, but also that loss of pylT does not compromise growth on other substrates. These results indicate that in‐depth genetic analysis of UAG translation as pyrrolysine is feasible, as deletion of pylT is conditionally lethal depending on growth substrate.

The acyl homoserine lactone receptor, SdiA, of Escherichia coli and Salmonella enterica serovar Typhimurium does not respond to indole.

ABSTRACT In this study, we tested the hypothesis that the SdiA proteins of Escherichia coli and Salmonella enterica serovar Typhimurium respond to indole. While indole was found to have effects on gene expression and biofilm formation, these effects were not sdiA dependent. However, high concentrations of indole did inhibit N-acyl-l-homoserine lactone (AHL) sensing by SdiA. We conclude that SdiA does not respond to indole but indole can inhibit SdiA activity in E. coli and Salmonella.

RamA, a Protein Required for Reductive Activation of Corrinoid-dependent Methylamine Methyltransferase Reactions in Methanogenic Archaea

Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function.