Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gastrointestinal tract (GIT) and vagina. Although not numerically dominant in the complex intestinal microflora, they are considered as key commensals that promote a healthy GIT. We determined the 2.26-Mb genome sequence of an infant-derived strain of Bifidobacterium longum, and identified 1,730 possible coding sequences organized in a 60%-GC circular chromosome. Bioinformatic analysis revealed several physiological traits that could partially explain the successful adaptation of this bacteria to the colon. An unexpectedly large number of the predicted proteins appeared to be specialized for catabolism of a variety of oligosaccharides, some possibly released by rare or novel glycosyl hydrolases acting on ''nondigestible'' plant polymers or host-derived glycoproteins and glycoconjugates. This ability to scavenge from a large variety of nutrients likely contributes to the competitiveness and persistence of bifidobacteria in the colon. Many genes for oligosaccharide metabolism were found in self-regulated modules that appear to have arisen in part from gene duplication or horizontal acquisition. Complete pathways for all amino acids, nucleotides, and some key vitamins were identified; however, routes for Asp and Cys were atypical. More importantly, genome analysis provided insights into the reciprocal interactions of bifidobacteria with their hosts. We identified polypeptides that showed homology to most major proteins needed for production of glycoprotein-binding fimbriae, structures that could possibly be important for adhesion and persistence in the GIT. We also found a eukaryotic-type serine protease inhibitor (serpin) possibly involved in the reported immunomodulatory activity of bifidobacteria.

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.Pseudomonas aeruginosa is a ubiquitous saprophyte and an opportunistic human pathogen which synthesizes numerous extracellular products including elastase, LasA protease, alkaline protease, phospholipase C, lipase, exotoxin A, rhamnolipids, hydrogen cyanide (HCN), and pyocyanin (25, 63). The production of these exoproducts, most of which can act as virulence factors, is positively controlled by two quorum-sensing signal molecules, N-(3-oxododecanoyl)-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-homoserine lactone (C4-HSL), which activate the transcription factors LasR and RhlR, respectively (20,26,59). The las and rhl systems are organized in a hierarchical manner such that the las system exerts transcriptional control over both rhlR and rhlI (26). A third signal molecule, 2-heptyl-3-hydroxy-4(1H)-quinolone, the synthesis and activity of which is linked to the las and rhl circuitry, is also required for virulence factor production and, in particular, for rhl-dependent exoproducts including pyocyanin and PA-IL lectin (41).Motility helps P. aeruginosa to colonize niches (10); three types of motility are observed, i.e., swimming, twitching, and swarming (24, 44). Whereas swimming in liquid media depends on flagella, twitching on solid media requires type IV pili.Swarming on semisolid media results from a combination of both types of motility and also requires rhamnolipid production (24, 46). Rhamnolipids are biosurfactants which not only enhance bacterial surface translocation by virtue of their wetting properties but also stimulate solubilization and degradation of hydrocarbons (35) and act as heat-stable hemolysins (21).The production of N-acyl-homoserine lactones (AHLs) and the expression of many virulence determinants in P. aeruginosa is negatively controlled at a posttranscriptional level by the small RNA-binding protein RsmA (42,43). This regulator is a homolog of CsrA in E...

The conserved two-component regulatory system GacS͞GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA-lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.translational control ͉ two-component regulatory system ͉ hydrogen cyanide ͉ biocontrol ͉ virulence