Dinoroseobacter shibae DFL12T , a member of the globally important marine Roseobacter clade, comprises symbionts of cosmopolitan marine microalgae, including toxic dinoflagellates. Its annotated 4 417 868 bp genome sequence revealed a possible advantage of this symbiosis for the algal host. D. shibae DFL12T is able to synthesize the vitamins B 1 and B 12 for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B 12 are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B 12 was confirmed to be functional, and D. shibae DFL12T was shown to provide the growth-limiting vitamins B 1 and B 12 to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12 T is able to grow anaerobically using the alternative electron acceptors nitrate and dimethylsulfoxide; it has the arginine deiminase survival fermentation pathway and a complex oxygen-dependent Fnr (fumarate and nitrate reduction) regulon. Many of these traits are shared with other members of the Roseobacter clade. D. shibae DFL12 T has five plasmids, showing examples for vertical recruitment of chromosomal genes (thiC) and horizontal gene transfer (cox genes, gene cluster of 47 kb) possibly by conjugation (vir gene cluster). The long-range (80%) synteny between two sister plasmids provides insights into the emergence of novel plasmids. D. shibae DFL12 T shows the most complex viral defense system of all Rhodobacterales sequenced to date.

Pseudomonas aeruginosa PAO1 produces the biodetergent rhamnolipid and secretes it into the extracellular environment. The role of rhamnolipids in the life cycle and pathogenicity of P. aeruginosa has not been completely understood, but they are known to affect outer membrane composition, cell motility, and biofilm formation. This report is focused on the influence of the outer membrane-bound esterase EstA of P. aeruginosa PAO1 on rhamnolipid production. EstA is an autotransporter protein which exposes its catalytically active esterase domain on the cell surface. Here we report that the overexpression of EstA in the wild-type background of P. aeruginosa PAO1 results in an increased production of rhamnolipids whereas an estA deletion mutant produced only marginal amounts of rhamnolipids. Also the known rhamnolipid-dependent cellular motility and biofilm formation were affected. Although only a dependence of swarming motility on rhamnolipids has been known so far, the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation. In order to demonstrate that EstA enzyme activity is responsible for these effects, inactive variant EstA* was constructed by replacement of the active serine by alanine. None of the mutant phenotypes could be complemented by expression of EstA*, demonstrating that the phenotypes affected by the estA mutation depend on the enzymatically active protein.Pseudomonas aeruginosa is an increasingly prevalent opportunistic gram-negative pathogen, which can infect humans, but also plants, nematodes, insects, amoebae, and animals (14,45,47). In humans, it causes infections in immunocompromised hosts such as patients suffering from cystic fibrosis, burns, or cancer and represents a major problem in intensive care units. P. aeruginosa produces and secretes an arsenal of virulence factors, which are all involved in the initiation or establishment of infection processes. These extracellular virulence determinants include not only proteases, lipases, and phospholipases but also nonprotein compounds like iron scavenger siderophores, the polysaccharide alginate, and the biosurfactant rhamnolipid, which is also known as heat-stable hemolysin (46).Many of these factors appear to have functions in complex physiological processes related to virulence like adhesion to different surfaces, cellular surface motility, and biofilm formation. P. aeruginosa is capable of performing three different types of cell motility: flagellum-mediated "swimming," type IV pilus-dependent "twitching," and a complex coordinated multicellular migration called "swarming" (16,39,48,54). In contrast to that of many other bacteria, swarming of P. aeruginosa is not dependent only on flagella but also on type IV pili (32). Furthermore, rhamnolipids seem to be required for swarming motility by acting as a surface-modifying agent (32).The rhamnolipids produced by P. aeruginosa are composed of mono-or dirhamnose linked to 3-hydroxy fatty acids of various chain lengths. The most abunda...

Pseudomonas aeruginosa is a human pathogen that frequently causes urinary tract and catheter-associated urinary tract infections. Here, using 13C-metabolic flux analysis, we conducted quantitative analysis of metabolic fluxes in the model strain P. aeruginosa PAO1 and 17 clinical isolates. All P. aeruginosa strains catabolized glucose through the Entner-Doudoroff pathway with fully respiratory metabolism and no overflow. Together with other NADPH supplying reactions, this high-flux pathway provided by far more NADPH than needed for anabolism: a benefit for the pathogen to counteract oxidative stress imposed by the host. P. aeruginosa recruited the pentose phosphate pathway exclusively for biosynthesis. In contrast to glycolytic metabolism, which was conserved among all isolates, the flux through pyruvate metabolism, the tricarboxylic acid cycle, and the glyoxylate shunt was highly variable, likely caused by adaptive processes in individual strains during infection. This aspect of metabolism was niche-specific with respect to the corresponding flux because strains isolated from the urinary tract clustered separately from those originating from catheter-associated infections. Interestingly, most glucose-grown strains exhibited significant flux through the glyoxylate shunt. Projection into the theoretical flux space, which was computed using elementary flux-mode analysis, indicated that P. aeruginosa metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response.

Pseudomonas aeruginosa secretes a variety of hydrolases, many of which contribute to virulence or are thought to play a role in the nutrition of the bacterium. As most studies concerning extracellular enzymes have been performed on planktonic cultures of non-mucoid P. aeruginosa strains, knowledge of the potential role of these enzymes in biofilm formation in mucoid (alginateproducing) P. aeruginosa remains limited. Here we show that mucoid P. aeruginosa produces extracellular hydrolases during biofilm growth. Overexpression of the extracellular lipases LipA and LipC, the esterase EstA and the proteolytic elastase LasB from plasmids revealed that some of these hydrolases affected the composition and physicochemical properties of the extracellular polymeric substances (EPS). While no influence of LipA was observed, the overexpression of estA and lasB led to increased concentrations of extracellular rhamnolipids with enhanced levels of mono-rhamnolipids, elevated amounts of total carbohydrates and decreased alginate concentrations, resulting in increased EPS hydrophobicity and viscosity. Moreover, we observed an influence of the enzymes on cellular motility. Overexpression of estA resulted in a loss of twitching motility, although it enhanced the ability to swim and swarm. The lasB-overexpression strain showed an overall enhanced motility compared with the parent strain. Moreover, the EstAand LasB-overproduction strains completely lost the ability to form 3D biofilms, whereas the overproduction of LipC increased cell aggregation and the heterogeneity of the biofilms formed. Overall, these findings indicate that directly or indirectly, the secreted enzymes EstA, LasB and LipC can influence the formation and architecture of mucoid P. aeruginosa biofilms as a result of changes in EPS composition and properties, as well as the motility of the cells. INTRODUCTIONPseudomonas aeruginosa is a common environmental bacterium and represents an increasingly prevalent opportunistic human pathogen whose ecological success is based on a remarkable degree of genomic flexibility and phenotypic adaptation (Wehmhöner et al., 2003). P. aeruginosa successfully colonizes a wide range of habitats, including natural soil and aquatic environments as well as artificial water systems (Botzenhart & Döring, 1993). It is involved in acute and chronic diseases, for example in infections of surgery and burn patients, in urogenital tract and wound infections, chronic lung infections of cystic fibrosis patients, and nosocomial pneumonia in intubated and mechanically ventilated patients (Drenkard, 2003;Singh et al., 2000).The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa under varying environmental conditions in nature, as well as in technical systems and the clinical setting. P. aeruginosa is now regarded as one of the medically most relevant biofilmforming bacterial species. Therefore, it has become one of the best-studied model organisms for biofilm formation (McDougald et al., 2008). Biofilm forma...

BackgroundLight, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime of the adduct state.ResultsHere, we report the photochemical, structural and functional characterization of DsLOV, a LOV protein from the photoheterotrophic marine α-proteobacterium Dinoroseobacter shibae which exhibits an average adduct state lifetime of 9.6 s at 20°C, and thus represents the fastest reverting bacterial LOV protein reported so far. Mutational analysis in D. shibae revealed a unique role of DsLOV in controlling the induction of photopigment synthesis in the absence of blue-light. The dark state crystal structure of DsLOV determined at 1.5 Å resolution reveals a conserved core domain with an extended N-terminal cap. The dimer interface in the crystal structure forms a unique network of hydrogen bonds involving residues of the N-terminus and the β-scaffold of the core domain. The structure of photoexcited DsLOV suggests increased flexibility in the N-cap region and a significant shift in the Cα backbone of β strands in the N- and C-terminal ends of the LOV core domain.ConclusionsThe results presented here cover the characterization of the unusual short LOV protein DsLOV from Dinoroseobacter shibae including its regulatory function, extremely fast dark recovery and an N-terminus mediated dimer interface. Due to its unique photophysical, structural and regulatory properties, DsLOV might thus serve as an alternative model system for studying light perception by LOV proteins and physiological responses in bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0365-0) contains supplementary material, which is available to authorized users.

Mucoid strains of Pseudomonas aeruginosa overproduce the exopolysaccharide alginate, which is substituted with O-acetyl groups. Under non-growing conditions in phosphate buffer, a mucoid clinical strain formed microcolonies on steel surfaces, while an acetylation-defective mutant was unable to form cell clusters. Enzymatic degradation of alginate by alginate lyase prevented microcolony formation of the mucoid parent strain. In a continuous-culture flow-cell system, using gluconate minimal medium, the mucoid strain with acetylated alginate formed microcolonies and grew into heterogenous biofilms, whereas the acetylation-defective mutant produced a thinner and more homogeneous biofilm. A lowered viscosity of extracellular material from the acetylation-defective mutant indicated a weakening of exopolymer interactions by loss of acetyl groups. These results suggest that acetyl substituents are necessary for the function of high-molecular-mass alginate to mediate cell aggregation into microcolonies in the early stages of biofilm development by mucoid P. aeruginosa, thereby determining the architecture of the mature biofilm.

Pseudomonas aeruginosa is a versatile opportunistic human pathogen that is able to colonize a broad spectrum of different aquatic and soil habitats. In the environment and during pathogenesis, P. aeruginosa encounters oxygen-limited and anaerobic environments. Particularly during chronic infection of the cystic fibrosis lung, oxygen-limiting conditions seem to contribute to persistent infection. Oxygen limitation increases antibiotic tolerance, robust biofilms and alginate biosynthesis, which contribute to the persistence of this opportunistic pathogen. Despite the importance of anaerobic metabolism during persistent infection of P. aeruginosa, we are just beginning to understand the underlying regulatory network and the molecular basis of how anaerobic metabolism contributes to a persistent infection. A deeper understanding of the anaerobic physiology of P. aeruginosa will allow the identification of new antibiotic targets and new therapeutic strategies.

BackgroundThe Roseobacter clade represents one of the most abundant, metabolically versatile and ecologically important bacterial groups found in marine habitats. A detailed molecular investigation of the regulatory and metabolic networks of these organisms is currently limited for many strains by missing suitable genetic tools.ResultsConjugation and electroporation methods for the efficient and stable genetic transformation of selected Roseobacter clade bacteria including Dinoroseobacter shibae, Oceanibulbus indolifex, Phaeobacter gallaeciensis, Phaeobacter inhibens, Roseobacter denitrificans and Roseobacter litoralis were tested. For this purpose an antibiotic resistance screening was performed and suitable genetic markers were selected. Based on these transformation protocols stably maintained plasmids were identified. A plasmid encoded oxygen-independent fluorescent system was established using the flavin mononucleotide-based fluorescent protein FbFP. Finally, a chromosomal gene knockout strategy was successfully employed for the inactivation of the anaerobic metabolism regulatory gene dnr from D. shibae DFL12T.ConclusionA genetic toolbox for members of the Roseobacter clade was established. This provides a solid methodical basis for the detailed elucidation of gene regulatory and metabolic networks underlying the ecological success of this group of marine bacteria.