Bacterial biofilms are highly dynamic communities which display a range of differentiated phenotypes during the course of development. By exchange of cell-cell signals, subpopulations of cells can coordinate their activity and undertake particular metabolic tasks or defense strategies (56). At times, the bacterial community releases single cells that escape from the biofilm and revert to a free-swimming, planktonic mode of growth, leaving behind hollow voids in the biofilm architecture (5, 37, 57). This process, referred to as dispersal, completes the biofilm life cycle and is thought to be important for successful colonization of new surfaces. Although the mechanisms underlying these events remain to be fully elucidated, previous studies of various species, including the opportunistic pathogen Pseudomonas aeruginosa, have revealed that dispersal events correlate with the induction of a specific phenotype that involves cellular motility (37, 42).In P. aeruginosa, biofilm dispersal can be triggered by environmental factors, including nutrient (42, 45) and iron (4, 36) availability, and has recently been linked to the intracellular second messenger cyclic di-GMP (c-di-GMP) (45, 47). Numerous studies revealed that decreased c-di-GMP levels are related to a motile mode of growth and to cell dispersal in eubacteria. In this second messenger system, diguanylate cyclases (DGCs) and specific phosphodiesterases (PDEs) are responsible for the biosynthesis and the degradation of c-di-GMP, respectively. DGCs and PDEs contribute to a genetic network that responds to a broad range of environmental cues and/or cell-cell signals and modulate intracellular levels of c-di-GMP, which has been shown to regulate various cellular functions, including biofilm formation, virulence, and dispersal, in many bacterial species (47,(51)(52)(53). Recently, we identified the gas nitric oxide (NO) as an important factor in the regulation of dispersal in P. aeruginosa biofilms (5). Exogenous addition of nontoxic concentrations of NO, typically in the low nanomolar range, was found to stimulate motility and biofilm dispersal in P. aeruginosa. A role for anaerobic metabolism and NO in biofilm dispersal and survival was further supported by other studies of P. aeruginosa (54, 61), Staphylococcus aureus (44), and various single and multispecies biofilms (6).NO is a water-soluble, hydrophobic free radical that can freely diffuse in biological systems. At high concentrations (micromolar to millimolar range), NO

In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation and dispersal of planktonic cellular aggregates, or ‘suspended biofilms’, by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10–400 µm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa.

The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679) exhibits enzyme activity on a range of substrates similar to that of its Ca2+-dependent counterpart PedE (PP_2674), including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La3+, Ce3+, Pr3+, Sm3+, or Nd3+. Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides.

Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a "sulfoglycolytic" pathway, in addition to the classical glycolytic (Embden-Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C 3 -organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C 3 -organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner-Doudoroff pathway for glucose-6-phosphate: It involves an NAD + -dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P) + -dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta-and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium.bacterial biodegradation | organosulfonate | sulfolipid | 6-deoxy-6-sulfoglucose | sulfur cycle S ulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipids sulfoquinovosyl diacylglycerols (SQDGs), which were discovered more than 60 y ago (1). The sulfolipids are located in the photosynthetic (thylakoid) membranes of all higher plants, mosses, ferns, and algae, as well as in most photosynthetic bacteria. They are also present in some nonphotosynthetic bacteria, and SQ is in the surface layer of some archaea (2-4). SQ is continuously degraded in all environments where SQ is produced, or it would enrich in these environments. The complete degradation of SQ to, ultimately, CO 2 , concomitant with a recycling of the bound sulfur in the form of inorganic sulfate, is dominated by microbes (e.g., by soil bacteria) (2, 5-9). However, a more detailed exploration of SQ-degrading microbes, of their SQ-degradation pathways, and of the enzymes and genes involved has only recently been initiated (10-12). The common view on SQ degradation is based on the consideration that SQ is a structural analog of glucose-6...

Ethylene glycol is used as a raw material in the production of polyethylene terephthalate, in antifreeze, as a gas hydrate inhibitor in pipelines, and for many other industrial applications. It is metabolized by aerobic microbial processes via the highly toxic intermediates glycolaldehyde and glycolate through C2 metabolic pathways. Pseudomonas putida KT2440, which has been engineered for environmental remediation applications given its high toxicity tolerance and broad substrate specificity, is not able to efficiently metabolize ethylene glycol, despite harboring putative genes for this purpose. To further expand the metabolic portfolio of P. putida, we elucidated the metabolic pathway to enable ethylene glycol via systematic overexpression of glyoxylate carboligase (gcl) in combination with other genes. Quantitative reverse transcription polymerase chain reaction demonstrated that all of the four genes in genomic proximity to gcl (hyi, glxR, ttuD, and pykF) are transcribed as an operon. Where the expression of only two genes (gcl and glxR) resulted in growth in ethylene glycol, improved growth and ethylene glycol utilization were observed when the entire gcl operon was expressed. Both glycolaldehyde and glyoxal inhibit growth in concentrations of ethylene glycol above 50 mM. To overcome this bottleneck, the additional overexpression of the glycolate oxidase (glcDEF) operon removes the glycolate bottleneck and minimizes the production of these toxic intermediates, permitting growth in up to 2 M (~124 g/L) and complete consumption of 0.5 M (31 g/L) ethylene glycol in shake flask experiments. In addition, the engineered strain enables conversion of ethylene glycol to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Overall, this study provides a robust P. putida KT2440 strain for ethylene glycol consumption, which will serve as a foundational strain for further biocatalyst development for applications in the remediation of waste polyester plastics and biomass-derived wastewater streams.

Cell aggregation is a stress response and serves as a survival strategy for Pseudomonas aeruginosa strain PAO1 during growth with the toxic detergent Na-dodecylsulfate (SDS). This process involves the psl operon and is linked to c-di-GMP signalling. The induction of cell aggregation in response to SDS was studied. Transposon and site-directed mutagenesis revealed that the cupA-operon and the co-transcribed genes siaA (PA0172) and siaD (PA0169) were essential for SDS-induced aggregation. While siaA encodes a putative membrane protein with a HAMP and a PP2C-like phosphatase domain, siaD encodes a putative diguanylate cyclase involved in the biosynthesis of c-di-GMP. Complementation studies uncovered that the loss of SDS-induced aggregation in the formerly isolated spontaneous mutant strain N was caused by a non-functional siaA allele. DNA-microarray analysis of SDS-grown cells revealed consistent activation of eight genes, including cupA1, with known or presumptive important functions in cell aggregation in the parent strain compared with non-aggregating siaA and siaD mutants. A siaAD-dependent increase of cupA1 mRNA levels in SDS-grown cells was also shown by Northern blots. These results clearly demonstrate that SiaAD are essential for inducing cell aggregation as a specific response to SDS and suggest that they are responsible for perceiving and transducing SDS-related stress.

Summary Pollution from ethylene glycol, and plastics containing this monomer, represent a significant environmental problem. The investigation of its microbial metabolism therefore provides insights into the environmental fate of this pollutant and also enables its utilization as a carbon source for microbial biotechnology. Here, we reveal the genomic and metabolic basis of ethylene glycol metabolism in Pseudomonas putida KT2440. Although this strain cannot grow on ethylene glycol as sole carbon source, it can be used to generate growth‐enhancing reducing equivalents upon co‐feeding with acetate. Mutants that utilize ethylene glycol as sole carbon source were isolated through adaptive laboratory evolution. Genomic analysis of these mutants revealed a central role of the transcriptional regulator GclR, which represses the glyoxylate carboligase pathway as part of a larger metabolic context of purine and allantoin metabolism. Secondary mutations in a transcriptional regulator encoded by PP_2046 and a porin encoded by PP_2662 further improved growth on ethylene glycol in evolved strains, likely by balancing fluxes through the initial oxidations of ethylene glycol to glyoxylate. With this knowledge, we reverse engineered an ethylene glycol utilizing strain and thus revealed the metabolic and regulatory basis that are essential for efficient ethylene glycol metabolism in P. putida KT2440.

Bile acids are surface-active steroid compounds with toxic effects for bacteria. Recently, the isolation and characterization of a bacterium, Pseudomonas sp. strain Chol1, growing with bile acids as the carbon and energy source was reported. In this study, initial reactions of the aerobic degradation pathway for the bile acid cholate were investigated on the biochemical and genetic level in strain Chol1. These reactions comprised A-ring oxidation, activation with coenzyme A (CoA), and ␤-oxidation of the acyl side chain with the C 19 -steroid dihydroxyandrostadienedione as the end product. A-ring oxidizing enzyme activities leading to ⌬ 1,4 -3-ketocholyl-CoA were detected in cell extracts and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cholate activation with CoA was demonstrated in cell extracts and confirmed with a chemically synthesized standard by LC-MS/MS. A transposon mutant with a block in oxidation of the acyl side chain accumulated a steroid compound in culture supernatants which was identified as 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) by nuclear magnetic resonance spectroscopy. The interrupted gene was identified as encoding a putative acyl-CoA-dehydrogenase (ACAD). DHOPDC activation with CoA in cell extracts of strain Chol1 was detected by LC-MS/MS. The growth defect of the transposon mutant could be complemented by the wild-type ACAD gene located on the plasmid pBBR1MCS-5. Based on these results, the initiating reactions of the cholate degradation pathway leading from cholate to dihydroxyandrostadienedione could be reconstructed. In addition, the first bacterial gene encoding an enzyme for a specific reaction step in side chain degradation of steroid compounds was identified, and it showed a high degree of similarity to genes in other steroid-degrading bacteria.