Phase-Variable Control of Multiple Phenotypes in Acinetobacter baumannii Strain AB5075

Abstract

Acinetobacter baumannii strain AB5075 produces colonies with two opacity phenotypes, designated opaque and translucent. These phenotypes were unstable and opaque and translucent colony variants were observed to interconvert at high frequency, suggesting that a phase-variable mechanism was responsible. The frequency of phase variation both within colonies and in broth cultures increased in a cell density-dependent manner and was mediated by the accumulation of an extracellular factor. This factor was distinct from the known A. baumannii signaling molecule 3-OH C 12 -homoserine lactone. Opaque and translucent colony variants exhibited a number of phenotypic differences, including cell morphology, surface motility, biofilm formation, antibiotic resistance, and virulence in a Galleria mellonella model. Additional clinical isolates exhibited a similar phase-variable control of colony opacity, suggesting that this may be a common feature of A. baumannii. IMPORTANCEA novel phase-variable mechanism has been identified in Acinetobacter baumannii that results in an interconversion between opaque and translucent colony phenotypes. This phase variation also coordinately regulates motility, cell shape, biofilm formation, antibiotic resistance, and virulence. The frequency of phase variation is increased at high cell density via a diffusible extracellular signal. To our knowledge, this report presents the first example of phase variation in A. baumannii and also the first example of quorum sensing-mediated control of phase variation in a bacterium. The findings are important, as this phase-variable mechanism can be identified only via changes in colony opacity using oblique light; therefore, many researchers studying A. baumannii may unknowingly be working with different colony variants.

An ompR-envZ Two-Component System Ortholog Regulates Phase Variation, Osmotic Tolerance, Motility, and Virulence in Acinetobacter baumannii Strain AB5075

“…Biofilm formation in E. coli is known to be regulated by many factors, including OmpR (29,30). The translucent variant of A. baumannii strain AB5075 produces more robust biofilms than the opaque variant on glass and polystyrene surfaces (10). Biofilm formation by the ΔompR, ΔenvZ, and ΔompR ΔenvZ mutant opaque and translucent variants was assessed, and no change in biofilm formation on polystyrene was apparent for any mutation in either the opaque or the translucent variant (see Fig.…”

“…Opaque variant colonies develop a sectored or mottled appearance when grown for 24 h or more at 37°C, which denotes phase switching from the opaque to the translucent variant (10). This visual indication was utilized as a marker to screen for transposon insertions that would increase sectoring after 24 h of incubation.…”

“…Although A. baumannii does not encode flagella, motility in this organism has been characterized (24)(25)(26)(27)(28). Furthermore, the opaque and translucent variants of A. baumannii strain AB5075 exhibit significant differences with regard to surface motility on soft agar plates (10). Since mutation of ompR and/or envZ yields increased phase variation from the opaque to translucent variant, motility by these mutants was assessed.…”

“…When cells are at high density, the colony variants interconvert at high frequency (1/10 1 to 1/10 2 ), indicating that an epigenetic mechanism is likely governing phase variation in A. baumannii. Also, omission of sodium chloride from growth medium had a negative effect on switching for both variants (O to T; T to O) (10).…”

“…Recently, a novel phase-variable mechanism was characterized that allows A. baumannii to interconvert between two phases or variants (opaque and translucent) that display markedly different phenotypes (10). Opaque variants (O) are more virulent and more motile and produce more 3-hydroxy-dodecanoyl-L-homoserine lactone (3-OH-C 12 -HSL) autoinducer, whereas the translucent variants (T) form more robust biofilms, are less virulent, and are less motile.…”