Autolysis and Autoaggregation in Pseudomonas aeruginosa Colony Morphology Mutants

Abstract

Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P. aeruginosa between survival of the many and persistence of the few.Pseudomonas aeruginosa, although readily defined taxonomically (69), shows no evidence of being constrained to a particular bacterial lifestyle (72). Indeed, this opportunistic pathogen proliferates within hosts as varied as plants, insects, nematodes, and mammals and interacts with amoebae and yeasts as well (13,16,31,44,58). The broad host range of P. aeruginosa can be used both to uncover the role of specific virulence factors and to characterize new regulatory systems controlling virulence (10,12,13,16,21,44,58). One study identified transposon insertion mutants of P. aeruginosa strain PAO1 that were less virulent in the fruit fly model host (16). Most such strains had an insertion in a chemotaxis-like regulatory locus (PA0408 to PA0417) required for surface spreading by twitching motility and controlling unknown factors required for efficient fly killing. Based only on their distinctive compact colony morphology, a large collection of these mutants was assembled for testing in the fly model.In this same study (16), two even more striking colony morphologies were noted, and strains of each type were saved. One set of mutants formed wrinkled colonies and had additional properties described for autoaggregating cells of Pseudomonas fluorescens (59,68) and Salmonella enterica serovar Typhimurium (61,62,83). The second set of mutants formed colonies with visible lysis, a characteristic noted in some of the earliest descriptions of P. aeruginosa isolates (5, 6, 26, 32), but for which the molecular basis has been elusive. Since autoaggregation and autolysis are likely to reflect fundamental aspects of the biology of P. aeruginosa, the genetic basis of the two mutant colony morphologies was explored. Autoaggregat...

Bacterial c-di-GMP Is an Immunostimulatory Molecule

“…The cyclic dinucleotide c-di-GMP is now recognized as a widespread intracellular signaling molecule in prokaryotes (2,3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). We have explored the extracellular biological effects of c-di-GMP and its potential use as a therapeutic agent.…”

“…The level of intracellular c-di-GMP in some species is believed to be regulated by the opposing effects of diguanylate cyclases for c-di-GMP synthesis from GTP and c-di-GMP phosphodiesterase for breakdown to 5Ј-GMP and each controlling the level of c-di-GMP in the cell. c-di-GMP has been shown to influence bacterial cell survival, cell differentiation, colonization and biofilm formation, as well as bacteria-host interactions (2,3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Although the role of intracellular c-di-GMP in bacteria is now recognized and is becoming an intense field of research, we have been studying the effect of this microbial molecule in the context of other biological systems and its potential clinical and therapeutic use for the prevention and inhibition of infection and disease (19 -22).…”

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation

“…Recently, a non-HSL P. aeruginosa intercellular signal, 2-heptyl 3-hydroxy 4-quinolone (PQS, Pseudomonas quinolone signal), a component of the P. aeruginosa quorum sensing hierarchy, has been described [19]. A chromosomal region encoding five proteins (PA0996 -1000) was recently shown to be essential for the production of PQS [30,31]. Four proteins encoded by this operon (PA0996 -PA0999) were synthesized at increased levels when P. aeruginosa was grown in low magnesium (Table 2 and Figure 1).…”

The structure–function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain

“…For example, PleD controls the onset of motility during the C. crescentus cell cycle (Aldridge et al, 2003;Paul et al, 2004). In Pseudomonas aeruginosa, autoaggregation is controlled by the GGDEF response regulator WspR (D'Argenio et al, 2002;Hickman et al, 2005), and twitching motility by FimX, which contains GGDEF and EAL domains (Huang et al, 2003;Kazmierczak et al, 2006). Additional examples of the effects of GGDEF domains on cell attachment and motility include ScrC regulation of attachment factors in Vibrio parahaemolyticus (Boles & McCarter, 2002), control of curli fimbriae by AdrA in S. typhimurium (Simm et al, 2004), and RocS effects on V. cholerae motility (Rashid et al, 2003).…”

“…A wspR : : mini-Tn5 WS mutant displays a smooth colony morphology (Spiers et al, 2002), and does not produce cellulose or attach to surfaces (Spiers et al, 2003). Expression of wspR in trans stimulates attachment and exopolysaccharide synthesis in various species (D'Argenio et al, 2002;Aldridge et al, 2003;Goymer et al, 2006;Ude et al, 2006). The evolutionary cause of LSWS is a single point mutation in the gene encoding the WspF methylesterase, which results in overactivation of the WspE kinase and constitutive activation of the primary output component of the Wsp pathway, WspR (Bantinaki et al, 2007).…”