Listeria monocytogenes is well known for its robust physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH. Although studies have provided insight into the mechanisms used by L. monocytogenes to allay the physiological consequences of these adverse environments, little is known about how these responses are coordinated. In the studies presented here, we have cloned the sigB gene and severalrsb genes from L. monocytogenes, encoding homologs of the alternative sigma factor ςB and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium Bacillus subtilis. TheL. monocytogenes and B. subtilis sigB andrsb genes are similar in sequence and physical organization; however, we observed that the activity of ςB in L. monocytogenes was uniquely responsive to osmotic upshifting, temperature downshifting, and the presence of EDTA in the growth medium. The magnitude of the response was greatest after an osmotic upshift, suggesting a role for ςB in coordinating osmotic responses in L. monocytogenes. A null mutation in the sigB gene led to substantial defects in the ability of L. monocytogenesto use betaine and carnitine as osmoprotectants. Subsequent measurements of betaine transport confirmed that the absence of ςB reduced the ability of the cells to accumulate betaine. Thus, ςB coordinates responses to a variety of physical and chemical signals, and its function facilitates the growth of L. monocytogenes under conditions of high osmotic strength.

Listeria monocytogenes is well known for its durable physiological characteristics, which allow the organism to grow at low temperature and pH and high osmolarity. Growth under high osmolarity depends on the accumulation of compatible solutes, among which glycine betaine and carnitine are the preferred solutes for this organism. Three different transport systems, Gbu, BetL, and OpuC, have been identified in L. monocytogenes which serve to scavenge the preferred compatible solutes. The general stress response regulator B has been shown to play an important role in osmotic adaptation in L. monocytogenes, presumably by directing transcription from one or more of the solute transport genes. In the studies presented here, we have used primer extension analyses to identify the promoter elements responsible for transcription of the opuC, gbuA, and betL genes. All three genes are osmotically inducible to some degree. betL is transcribed from a Bindependent promoter, while gbuA is transcribed from dual promoters, one of which is B dependent. opuC is transcribed exclusively from a B -dependent promoter. The betL promoter is similar in sequence to the B -independent gbuAP1 promoter. Kinetic analysis of transcript accumulation after osmotic upshift demonstrated that B -dependent transcripts from gbuAP2 and sigB accumulate for an extended period after upshift, suggesting that B activity may provide a mechanism for sustained high-level expression during osmotic challenge. In contrast to osmotic upshift, expression from the B -dependent opuC and gbuAP2 promoters after temperature upshift and ethanol stress was minimal, suggesting that additional mechanisms may also participate in regulating transcription from these B -dependent promoters.

K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10th domain of human fibronectin as their scaffold, are stable inside the cells and when fused with a fluorescent protein label, the constitutively active G12V mutant H-Ras. Primary selection of ligands against Ras with mRNA display resulted in an intrabody (termed RasIn1) that binds with a KD of 2.1 μM to H-Ras(G12V) (GTP), excellent state selectivity, and remarkable specificity for K- and H-Ras. RasIn1 recognizes residues in the Switch I region of Ras, similar to Raf-RBD, and competes with Raf-RBD for binding. An affinity maturation selection based on RasIn1 resulted in RasIn2, which binds with a KD of 120 nM and also retains excellent state selectivity. Both of these intrabodies colocalize with H-Ras, K-Ras, and G12V mutants inside the cells, providing new potential tools to monitor and modulate Ras-mediated signaling. Finally, RasIn1 and Rasin2 both display selectivity for the G12V mutants as compared with wild-type Ras providing a potential route for mutant selective recognition of Ras.