The Gram-negative pathogen Acinetobacter baumannii strain M2 was found to exhibit a robust surface motility on low-percentage (0.2-0.4 %) agar plates. These patterns of motility were dramatically different depending on whether Difco or Eiken agar was used. Motility was observed in many, but not all, clinical and environmental isolates. The use of drop collapse assays to demonstrate surfactant production was unsuccessful, and the role of surfactants in A. baumannii M2 motility remains unclear. Surface motility was impaired by an insertion in pilT, encoding a gene product that is often required for retraction of the type IV pilus. Motility was also dependent on quorum sensing, as a null allele in the abaI autoinducer synthase decreased motility, and the addition of exogenous N-(3-hydroxy)-dodecanoylhomoserine lactone (3-OH C 12 -HSL) restored motility to the abaI mutant. Transposon mutagenesis was used to identify additional genes required for motility and revealed loci encoding various functions: non-ribosomal synthesis of a putative lipopeptide, a sensor kinase (BfmS), a lytic transglycosylase, O-antigen biosynthesis (RmlB), an outer membrane porin (OmpA) and de novo purine biosynthesis (PurK). Two of the above genes required for motility were highly activated by quorum sensing, and may explain, in part, the requirement for quorum sensing in motility.

The opportunistic human pathogen Acinetobacter baumannii strain M2 was found to produce distinct acyl-homoserine lactone (AHL) signals based on the use of an Agrobacterium tumefaciens traG-lacZ biosensor. An A. baumannii gene, designated abaI, was cloned and directed AHL production in recombinant Escherichia coli. The AbaI protein was similar to members of the LuxI family of autoinducer synthases and was predicted to be the only autoinducer synthase encoded by A. baumannii. The primary AHL signal directed by AbaI was identified by mass spectrometry as being N-(3-hydroxydodecanoyl)-L-HSL (3-hydroxy-C 12 -HSL). Minor amounts of at least five additional AHLs were also identified. The expression of abaI at the transcriptional level was activated by ethyl acetate extracts of culture supernatants or by synthetic 3-hydroxy-C 12 -HSL. An abaI::Km mutant failed to produce any detectable AHL signals and was impaired in biofilm development.

The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and is required for the production of an unknown quorum-sensing molecule. In a screen to identify rhomboid-encoding genes from Proteus mirabilis, tatA was identified as a multicopy suppressor and restored extracellular signal production as well as complementing all other phenotypes of a Prov. stuartii aarA mutant. TatA is a component of the twin-arginine translocase (Tat) protein secretion pathway and likely forms a secretion pore. By contrast, the native tatA gene of Prov. stuartii in multicopy did not suppress an aarA mutation. We find that TatA in Prov. stuartii has a short N-terminal extension that was atypical of TatA proteins from most other bacteria. This extension was proteolytically removed by AarA both in vivo and in vitro. A Prov. stuartii TatA protein missing the first 7 aa restored the ability to rescue the aarA-dependent phenotypes. To verify that loss of the Tat system was responsible for the various phenotypes exhibited by an aarA mutant, a tatC-null allele was constructed. The tatC mutant exhibited the same phenotypes as an aarA mutant and was epistatic to aarA. These data provide a molecular explanation for the requirement of AarA in quorum-sensing and uncover a function for the Tat protein export system in the production of secreted signaling molecules. Finally, TatA represents a validated natural substrate for a prokaryotic rhomboid protease.

No abstract

Proteus mirabilis is a Gram-negative bacterium that undergoes a physical and biochemical change from a vegetative swimmer cell (a typical Gram-negative rod) to an elongated swarmer cell when grown on a solid surface. In this study, we report that a transposon insertion in the waaL gene, encoding O-antigen ligase, blocked swarming motility on solid surfaces but had little effect on swimming motility in soft agar. The waaL mutant was unable to differentiate into a swarmer cell. Differentiation was also prevented by a mutation in wzz, encoding a chain length determinant for O antigen, but not by a mutation in wzyE, encoding an enzyme that polymerizes enterobacterial common antigen, a surface polysaccharide different from the lipid A::core. In wild-type P. mirabilis, increased expression of the flhDC operon occurs after growth on solid surfaces and is required for the high-level expression of flagellin that is characteristic of swarmer cells. However, in both the waaL and the wzz mutants, the flhDC operon was not activated during growth on agar. A loss-of-function mutation in the rcsB response regulator or overexpression of flhDC restored swarming to the waaL mutant, despite the absence of O antigen. Therefore, although O antigen may serve a role in swarming by promoting wettability, the loss of O antigen blocks a regulatory pathway that links surface contact with the upregulation of flhDC expression.