RpoS, the sigma factor of enteric bacteria that responds to stress and stationary phase, is subject to complex regulation acting at multiple levels, including transcription, translation, and proteolysis. Increased translation of rpoS mRNA during growth at low temperature, after osmotic challenge, or with a constitutively activated Rcs phosphorelay depends on two trans-acting small regulatory RNAs (sRNAs) in Escherichia coli. The DsrA and RprA sRNAs are both highly conserved in Salmonella enterica, as is their target, an inhibitory antisense element within the rpoS untranslated leader. Analysis of dsrA and rprA deletion mutants indicates that while the increased translation of RpoS in response to osmotic challenge is conserved in S. enterica, dependence on these two sRNA regulators is much reduced. Furthermore, low-temperature growth or constitutive RcsC activation had only modest effects on RpoS expression, and these increases were, respectively, independent of dsrA or rprA function. This lack of conservation of sRNA function suggests surprising flexibility in RpoS regulation.RpoS, the general stress and stationary-phase (SP) sigma factor, is highly conserved among Escherichia coli, Salmonella enterica, and other related enteric bacteria. The diverse and often harsh conditions encountered by these bacteria, whether residing as pathogens in the gut or as saprophytes in the environment, require the ability to integrate multiple stress signals and initiate the appropriate cellular responses in order to survive. RpoS serves in this capacity as the master regulator of the general stress response. Its levels increase in response to a number of stress signals, including osmotic shock, nutrient depletion, low temperature, and growth into stationary phase (reviewed in reference 19). As RpoS becomes more abundant, it effectively competes with the vegetative sigma factor in binding to core RNA polymerase, leading to increased transcription of genes necessary for mediating the stress response (49).Regulation of RpoS is complex, with a large posttranscriptional component, and involves trans-acting factors (19). These factors include several small regulatory RNAs (28, 39) which target a cis-acting antisense element within the rpoS mRNA untranslated leader (7). In E. coli, two such small RNAs (sRNAs), DsrA and RprA, activate rpoS translation by binding to and inhibiting the antisense element (reviewed in reference 30). DsrA is necessary for activation of rpoS translation in response to low temperature and osmotic shock (27), while RprA increases RpoS both in response to osmotic shock (29) and in response to a constitutively active rcsC allele, indicating a role in cell envelope stress (15,29).These sRNAs were initially discovered and characterized in E. coli, and their gene sequences are Ϸ90% identical in S. enterica. The high degree of sequence conservation shared by E. coli and S. enterica, in both rpoS and the sRNAs, suggested that their regulatory functions are likely to be conserved as well. Here we describe the results o...

The sequence/function space in the d-mannonate dehydratase subgroup (ManD) of the enolase superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry53, 2722–2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 103–104 M–1 s–1) for dehydration of d-mannonate, low-efficiency (kcat/KM = 10–102 M–1 s–1) for dehydration of d-mannonate and/or d-gluconate, and no activity. Characterization of high-efficiency members revealed that these are ManDs in the d-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087–4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for l-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for l-gulonate catabolism in Escherichia coli CFT073 and l-idonate catabolism in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109. However, we could not identify physiological roles for the low-efficiency members of the ManD subgroup, allowing the suggestion that these pathways may be either evolutionary relics or the starting points for new metabolic potential.

Archaea, plants, and most bacteria synthesize heme using the C5 pathway, in which the first committed step is catalyzed by the enzyme glutamyl-tRNA reductase (GluTR or HemA). In some cases, an overproduced and purified HemA enzyme contains noncovalently bound heme. The enteric bacteria Salmonella enterica and Escherichia coli also synthesize heme by the C5 pathway, and the HemA protein in these bacteria is regulated by proteolysis. The enzyme is unstable during normal growth due to the action of Lon and ClpAP, but becomes stable when heme is limiting for growth. We describe a method for the overproduction of S. enterica HemA that yields a purified enzyme containing bound heme, identified as a b-type heme by spectroscopy. A mutant of HemA (C170A) does not contain heme when similarly purified. The mutant was used to test whether heme is directly involved in HemA regulation. When expressed from the S. enterica chromosome in a wild-type background, the C170A mutant allele of hemA is shown to confer an unregulated phenotype, with high levels of HemA regardless of the heme status. These results strongly suggest that the presence of bound heme targets the HemA enzyme for degradation and is required for normal regulation.

The first part of this thesis is dedicated to translational regulation of rpoS mRNA by the small noncoding RNAs (sRNAs), DsrA and RprA, in two closely related enteric bacteria, Escherichia coli, and Salmonella enterica serovar Typhimurium. The rpoS gene encodes a second vegetative sigma factor for RNA polymerase, which directs the cell's transcriptional response to general stress and entry into stationary phase. The rpoS gene is highly conserved among the γ−branch of proteobacteria, and sRNAs are highly conserved in related species. In fact, sequence conservation is thought to have predictive value in sRNA discovery and functional conservation is largely assumed. First discovered in E. coli, DsrA and RprA were shown to activate rpoS translation in response to low temperature and osmotic shock respectively. Base pairing between these sRNAs and rpoS mRNA disrupts a hairpin in the untranslated leader region of rpoS that blocks ribosome binding. The function of these sRNAs was tested in S. enterica serovar Typhimurium under the same conditions reported to be important for their function in E. coli. Neither DsrA nor RprA was required for rpoS regulation in S. enterica. Importantly, this work demonstrates that sRNA function cannot be inferred from sequence conservation. thanks to Andrew Hiss and Maret Bernard, both of whom never failed to lend me a shoulder, a couch, and a wireless connection. Thanks also to Adrian Larry for providing the soundtrack.