Genetic tools for the investigation of Roseobacter clade bacteria

<p>Abstract</p> <p>Background</p> <p>The <it>Roseobacter </it>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.</p> <p>Results</p> <p>Conjugation and electroporation methods for the efficient and stable genetic transformation of selected <it>Roseobacter </it>clade bacteria including <it>Dinoroseobacter shibae</it>, <it>Oceanibulbus indolifex</it>, <it>Phaeobacter gallaeciensis</it>, <it>Phaeobacter inhibens</it>, <it>Roseobacter denitrificans </it>and <it>Roseobacter litoralis </it>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 <it>dnr </it>from <it>D. shibae </it>DFL12^T.</p> <p>Conclusion</p> <p>A genetic toolbox for members of the <it>Roseobacter </it>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.</p

Added Value of Molecular Biological Analysis in Diagnosis and Clinical Management of Liposarcoma: A 30-Year Single-Institution Experience

Biological, physical and clinical aspects of cancer treatment with ionising radiatio

Influence of extracellular enzymes on the structure and properties of biofilms of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an increasingly prevalent opportunistic human pathogen. It secretes a variety of hydrolases, many of which contribute to virulence or are thought to play a role in nutrition of the bacterium. However, potential roles of these extracellular enzymes in biofilm formation or composition of the extracellular polymeric substances (EPS) have not been studied. The aim of this study was to compare biofilms of the mucoid P. aeruginosa strain SG81 and its derivatives overexpressing different extracellular hydrolases. For this the genes for lipases LipA and LipC, the esterase EstA and elastase LasB were moderately overexpressed from plasmids in strain SG81. Biofilms were grown in flow-cells, analyzed by confocal laser-scanning microscope (CLSM) and the effects on EPS composition and physicochemical properties were investigated. Whereas lipA overexpression revealed no significant differences, the other enzymes influenced biofilm structure and/or EPS properties. In contrast to the parent strain, cells overproducing LipC were more heterogeniously distributed in the biofilm which was predominently composed of microcolonies consisting of highly packed cells. However, LipC overproduction had no influence on the EPS composition. Overproduction of EstA led to differences in biofilm architecture and resulted in premature biofilm dissolution. Compared to the parent strain the EPS showed differences in the concentrations of carbohydrates and rhamnolipids. Moreover, hydrophobicity and viscosity of the EPS were significantly increased. Overproduction of LasB had the most pronounced effects on biofilms: a drastically reduced ability to form an elaborate biofilm architecture resulting in a monolayer of unevenly distributed cells even after prolonged times of growth. Furthermore, the concentration of alginate was reduced, whereas the total concentration of carbohydrates increased. Like in the EstA overproducing strain the hydrophobicity and viscosity of the EPS were increased. With Gfp-enzympromotor-fusions it was possible to located a continuous transcription of lipA in all cells of the biofilm. In Contrast the transcription activity of the estA gene occurred only in a few cells at a later state of biofilm developement. These results clearly indicate that overexpression of extracellular enzymes can affect EPS composition and properties, thereby influencing biofilm development. Altogether, this data provides strong evidence that extracellular enzymes, in addition to their roles in virulence and nutrition, have a key role in the differentiation of microbial biofilms

Genome Sequence of the Acute Urethral Catheter Isolate Pseudomonas aeruginosa MH38

Wibberg D, Tielen P, Blom J, et al. Genome Sequence of the Acute Urethral Catheter Isolate Pseudomonas aeruginosa MH38. Genome announcements. 2014;2(2): e00161-14.Pseudomonas aeruginosa is a major nosocomial bacterial pathogen causing complicated catheter-associated urinary tract infections (CAUTIs). Here, we present the 6.9-Mb draft genome sequence of P. aeruginosa MH38 isolated from an acute nosocomial CAUTI. It exhibits resistance to several antibiotics but revealed low-level production of virulence factors

Robustness and Plasticity of Metabolic Pathway Flux among Uropathogenic Isolates of <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa</i> 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 <i>P. aeruginosa</i> PAO1 and 17 clinical isolates. All <i>P. aeruginosa</i> 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. <i>P. aeruginosa</i> 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 <i>P. aeruginosa</i> metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response.</p></div

Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae

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

Gene Flow Across Genus Barriers - Conjugation of Dinoroseobacter shibae's 191-kb Killer Plasmid into Phaeobacter inhibens and AHL-mediated Expression of Type IV Secretion Systems.

Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore the exchange of genetic material via horizontal transfer has been supposed to be a major evolutionary driving force. Many plasmids in this group encode type IV secretion systems (T4SS) that are expected to mediate transfer of proteins and/or DNA into host cells, but no experimental evidence of either has yet been provided. Dinoroseobacter shibae, a species of the Roseobacter group within the Rhodobacteraceae family, contains five ECRs that are crucial for anaerobic growth, survival under starvation and the pathogenicity of this model organism. Here we tagged two syntenous but compatible RepABC-type plasmids of 191 and 126-kb size, each encoding a T4SS, with antibiotic resistance genes and demonstrated their conjugational transfer into a distantly related Roseobacter species, namely Phaeobacter inhibens. Pulsed field gel electrophoresis showed transfer of those replicons into the recipient both individually but also together documenting the efficiency of conjugation. We then studied the influence of externally added quorum sensing (QS) signals on the expression of the T4SS located on the sister plasmids. A QS deficient D. shibae null mutant (ΔluxI1 ) lacking synthesis of N-acyl-homoserine lactones (AHLs) was cultivated with a wide spectrum of chemically diverse long-chain AHLs. All AHLs with lengths of the acid side-chain ≥14 reverted the ΔluxI1 phenotype to wild-type. Expression of the T4SS was induced up to log2 ∼3fold above wild-type level. We hypothesize that conjugation in roseobacters is QS-controlled and that the QS system may detect a wide array of long-chain AHLs at the cell surface