The outstanding metabolic and bioprotective properties of the bacterial genusPseudomonasmake thes... more The outstanding metabolic and bioprotective properties of the bacterial genusPseudomonasmake these species a potentially interesting source for the search of hydrolytic activities that could be useful for the degradation of plastics. We identified two genes encoding the intracellular lipases LIP1 and LIP2 of the biocontrol bacteriumPseudomonas chlororaphisPA23 and subsequently performed cloning and expression inEscherichia coli. Thelip1gene has an open reading frame of 828 bp and encodes a protein of 29.7 kDa whereas thelip2consists of 834 bp and has a protein of 30.2 kDa. Although secondary structure analyses of LIP1 and LIP2 indicate a dominant α/β-hydrolase-fold, the two proteins differ widely in their amino acid sequences (15.39% identity), substrate specificities, and hydrolysis rates. Homology modeling indicates the catalytic serine in both enzymes located in a GXSXG sequence motif (lipase box). However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with a GGX-type oxyani...
Synthetic plastics are pivotal in our current lifestyle and therefore, its accumulation is a majo... more Synthetic plastics are pivotal in our current lifestyle and therefore, its accumulation is a major concern for environment and human health. Petroleum-derived (petro-)polymers such as polyethylene (PE), polyethylene terephthalate (PET), polyurethane (PU), polystyrene (PS), polypropylene (PP), and polyvinyl chloride (PVC) are extremely recalcitrant to natural biodegradation pathways. Some microorganisms with the ability to degrade petro-polymers under in vitro conditions have been isolated and characterized. In some cases, the enzymes expressed by these microbes have been cloned and sequenced. The rate of polymer biodegradation depends on several factors including chemical structures, molecular weights, and degrees of crystallinity. Polymers are large molecules having both regular crystals (crystalline region) and irregular groups (amorphous region), where the latter provides polymers with flexibility. Highly crystalline polymers like polyethylene (95%), are rigid with a low capacity...
Polyethylene (PE) is the most abundant synthetic, petroleum-based plastic materials produced glob... more Polyethylene (PE) is the most abundant synthetic, petroleum-based plastic materials produced globally, and one of the most resistant to biodegradation, resulting in massive accumulation in the environment. Although the microbial degradation of polyethylene has been reported, complete biodegradation of polyethylene has not been achieved, and rapid degradation of polyethylene under ambient conditions in the environment is still not feasible. Experiments reported in the literature suffer from a number of limitations, and conclusive evidence for the complete biodegradation of polyethylene by microorganisms has been elusive. These limitations include the lack of a working definition for the biodegradation of polyethylene that can lead to testable hypotheses, a non-uniform description of experimental conditions used, and variations in the type(s) of polyethylene used, leading to a profound limitation in our understanding of the processes and mechanisms involved in the microbial degradatio...
The rheology of high-cell density (HCD) cultures is an important parameter for its impact on mixi... more The rheology of high-cell density (HCD) cultures is an important parameter for its impact on mixing and sparging, process scale-up, and downstream unit operations in bioprocess development. In this work, time-dependent rheological properties of HCD Pseudomonas putida LS46 cultures were monitored for microbial polyhydroxyalkanoate (PHA) production. As the cell density of the fed-batch cultivation increased (0 to 25 g·L−1 cell dry mass, CDM), the apparent viscosity increased nearly nine-fold throughout the fed-batch process. The medium behaved as a nearly Newtonian fluid at lower cell densities, and became increasingly shear-thinning as the cell density increased. However, shear-thickening behavior was observed at shearing rates of approximately 75 rad·s−1 or higher, and its onset increased with viscosity of the sample. The supernatant, which contained up to 9 g·L−1 soluble organic material, contributed more to the observed viscosity effect than did the presence of cells. Owing to thi...
Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a b... more Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay’s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and ...
Microbial polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that may alleviate so... more Microbial polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that may alleviate some of the environmental burden of petroleum-derived polymers. The requirements for carbon substrates and energy for bioreactor operations are major factors contributing to the high production costs and environmental impact of PHAs. Improving the process productivity is an important aspect of cost reduction, which has been attempted using a variety of fed-batch, continuous, and semi-continuous bioreactor systems, with variable results. The purpose of this review is to summarize the bioreactor operations targeting high PHA productivity using pure cultures. The highest volumetric PHA productivity was reported more than 20 years ago for poly(3-hydroxybutryate) (PHB) production from sucrose (5.1 g L−1 h−1). In the time since, similar results have not been achieved on a scale of more than 100 L. More recently, a number fed-batch and semi-continuous (cyclic) bioreactor operation strategies have...
Six secretion systems (Type I–Type VI) have been reported in Gram-negative bacteria. The Type VI ... more Six secretion systems (Type I–Type VI) have been reported in Gram-negative bacteria. The Type VI secretion (T6SS) system was identified for the first time in two human pathogens, and its components assemble a system for secretion of the hemolysin-coregulated protein (Hcp1) and the valine glycine repeat protein (VgrG). Putative genes encoding T6SS are present not only in bacteria identified as human or plant pathogens but also in symbiotic nitrogen-fixing bacteria and nonpathogenic soil bacteria. Pseudomonas putida LS46 was isolated from wastewater and was identified as a producer of medium chain length polyhydroxyalkanoates (mcl-PHAs), natural polyester polymers synthesized by bacteria as energy storage molecules. Using the COG ID of the T6SS from P. aerugonosa PAO1 as a probe, three putative T6SSs were identified in P. putida LS46. Two of the T6SSs (S1 and S2) were identical in their organization, and their gene products showed a high degree of amino acid sequence homology. The third T6SS (S3) had a different gene organization, and some of the T6SS genes present in S1/S2 were absent in S3. Many P. putida strains have highly conserved T6SS genes, which have low amino acid sequence identity to T6SS proteins of Vibrio cholerae, Aeromonas hydrophila, Rhizobium leguminosarum, and Burkholderia mallei. Accumulation of PHA in bacteria helps to withstand starvation and survive under hostile environmental conditions. We speculate the GacA/GacS system regulates Quorum Sensing, which in turn regulates T6SS. T6SS effector molecules could be involved in signal transduction within the population for induction of PHA synthesis and accumulation.
A bacterium, isolated from wastewater after enrichment with waste canola fryer oil, was found to ... more A bacterium, isolated from wastewater after enrichment with waste canola fryer oil, was found to synthesize 12–20 % of cell dry weight (cdw) medium chain length polyhydroxyalkanoates (mcl-PHAs) using different carbon substrates. On the basis of partial 16S rDNA sequence analysis, this bacterium was first identified as Pseudomonas putida and designated as P. putida strain MO2. However, the full 16S rDNA gene sequence from a whole genome sequence analysis of this strain showed 100 % identity to 16S rDNA of Pseudomonas monteilii SB3101 and Pseudomonas monteilii SB3078. The comparison of the cpn60 gene sequence of Pseudomonas sp. strain MO2 with strains of P. putida, P. aeruginosa, P. fluorescens, P. stutzeri, P. syringae, and P. monteilii indicated that this strain is more closely related to P. monteilii than P. putida type strain KT2440. Based on gene sequence similarity index and phylogenetic analyses, some P. putida strains, which were earlier classified as P. putida, are also more closely related to the P. monteilii cluster. Our analyses show that P. putida is a very diverse group with divergent strains, and many strains of P. putida that cluster with P. monteilii species may need to be reclassified.
Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is... more Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal int...
We describe the draft genome sequence of Pseudomonas putida strain LS46, a novel isolate that syn... more We describe the draft genome sequence of Pseudomonas putida strain LS46, a novel isolate that synthesizes medium-chain-length polyhydroxyalkanoates. The draft genome of P. putida LS46 consists of approximately 5.86 million bp, with a G+C content of 61.69%. A total of 5,316 annotated genes and 5,219 coding sequences (CDS) were identified.
The outstanding metabolic and bioprotective properties of the bacterial genusPseudomonasmake thes... more The outstanding metabolic and bioprotective properties of the bacterial genusPseudomonasmake these species a potentially interesting source for the search of hydrolytic activities that could be useful for the degradation of plastics. We identified two genes encoding the intracellular lipases LIP1 and LIP2 of the biocontrol bacteriumPseudomonas chlororaphisPA23 and subsequently performed cloning and expression inEscherichia coli. Thelip1gene has an open reading frame of 828 bp and encodes a protein of 29.7 kDa whereas thelip2consists of 834 bp and has a protein of 30.2 kDa. Although secondary structure analyses of LIP1 and LIP2 indicate a dominant α/β-hydrolase-fold, the two proteins differ widely in their amino acid sequences (15.39% identity), substrate specificities, and hydrolysis rates. Homology modeling indicates the catalytic serine in both enzymes located in a GXSXG sequence motif (lipase box). However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with a GGX-type oxyani...
Synthetic plastics are pivotal in our current lifestyle and therefore, its accumulation is a majo... more Synthetic plastics are pivotal in our current lifestyle and therefore, its accumulation is a major concern for environment and human health. Petroleum-derived (petro-)polymers such as polyethylene (PE), polyethylene terephthalate (PET), polyurethane (PU), polystyrene (PS), polypropylene (PP), and polyvinyl chloride (PVC) are extremely recalcitrant to natural biodegradation pathways. Some microorganisms with the ability to degrade petro-polymers under in vitro conditions have been isolated and characterized. In some cases, the enzymes expressed by these microbes have been cloned and sequenced. The rate of polymer biodegradation depends on several factors including chemical structures, molecular weights, and degrees of crystallinity. Polymers are large molecules having both regular crystals (crystalline region) and irregular groups (amorphous region), where the latter provides polymers with flexibility. Highly crystalline polymers like polyethylene (95%), are rigid with a low capacity...
Polyethylene (PE) is the most abundant synthetic, petroleum-based plastic materials produced glob... more Polyethylene (PE) is the most abundant synthetic, petroleum-based plastic materials produced globally, and one of the most resistant to biodegradation, resulting in massive accumulation in the environment. Although the microbial degradation of polyethylene has been reported, complete biodegradation of polyethylene has not been achieved, and rapid degradation of polyethylene under ambient conditions in the environment is still not feasible. Experiments reported in the literature suffer from a number of limitations, and conclusive evidence for the complete biodegradation of polyethylene by microorganisms has been elusive. These limitations include the lack of a working definition for the biodegradation of polyethylene that can lead to testable hypotheses, a non-uniform description of experimental conditions used, and variations in the type(s) of polyethylene used, leading to a profound limitation in our understanding of the processes and mechanisms involved in the microbial degradatio...
The rheology of high-cell density (HCD) cultures is an important parameter for its impact on mixi... more The rheology of high-cell density (HCD) cultures is an important parameter for its impact on mixing and sparging, process scale-up, and downstream unit operations in bioprocess development. In this work, time-dependent rheological properties of HCD Pseudomonas putida LS46 cultures were monitored for microbial polyhydroxyalkanoate (PHA) production. As the cell density of the fed-batch cultivation increased (0 to 25 g·L−1 cell dry mass, CDM), the apparent viscosity increased nearly nine-fold throughout the fed-batch process. The medium behaved as a nearly Newtonian fluid at lower cell densities, and became increasingly shear-thinning as the cell density increased. However, shear-thickening behavior was observed at shearing rates of approximately 75 rad·s−1 or higher, and its onset increased with viscosity of the sample. The supernatant, which contained up to 9 g·L−1 soluble organic material, contributed more to the observed viscosity effect than did the presence of cells. Owing to thi...
Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a b... more Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay’s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and ...
Microbial polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that may alleviate so... more Microbial polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that may alleviate some of the environmental burden of petroleum-derived polymers. The requirements for carbon substrates and energy for bioreactor operations are major factors contributing to the high production costs and environmental impact of PHAs. Improving the process productivity is an important aspect of cost reduction, which has been attempted using a variety of fed-batch, continuous, and semi-continuous bioreactor systems, with variable results. The purpose of this review is to summarize the bioreactor operations targeting high PHA productivity using pure cultures. The highest volumetric PHA productivity was reported more than 20 years ago for poly(3-hydroxybutryate) (PHB) production from sucrose (5.1 g L−1 h−1). In the time since, similar results have not been achieved on a scale of more than 100 L. More recently, a number fed-batch and semi-continuous (cyclic) bioreactor operation strategies have...
Six secretion systems (Type I–Type VI) have been reported in Gram-negative bacteria. The Type VI ... more Six secretion systems (Type I–Type VI) have been reported in Gram-negative bacteria. The Type VI secretion (T6SS) system was identified for the first time in two human pathogens, and its components assemble a system for secretion of the hemolysin-coregulated protein (Hcp1) and the valine glycine repeat protein (VgrG). Putative genes encoding T6SS are present not only in bacteria identified as human or plant pathogens but also in symbiotic nitrogen-fixing bacteria and nonpathogenic soil bacteria. Pseudomonas putida LS46 was isolated from wastewater and was identified as a producer of medium chain length polyhydroxyalkanoates (mcl-PHAs), natural polyester polymers synthesized by bacteria as energy storage molecules. Using the COG ID of the T6SS from P. aerugonosa PAO1 as a probe, three putative T6SSs were identified in P. putida LS46. Two of the T6SSs (S1 and S2) were identical in their organization, and their gene products showed a high degree of amino acid sequence homology. The third T6SS (S3) had a different gene organization, and some of the T6SS genes present in S1/S2 were absent in S3. Many P. putida strains have highly conserved T6SS genes, which have low amino acid sequence identity to T6SS proteins of Vibrio cholerae, Aeromonas hydrophila, Rhizobium leguminosarum, and Burkholderia mallei. Accumulation of PHA in bacteria helps to withstand starvation and survive under hostile environmental conditions. We speculate the GacA/GacS system regulates Quorum Sensing, which in turn regulates T6SS. T6SS effector molecules could be involved in signal transduction within the population for induction of PHA synthesis and accumulation.
A bacterium, isolated from wastewater after enrichment with waste canola fryer oil, was found to ... more A bacterium, isolated from wastewater after enrichment with waste canola fryer oil, was found to synthesize 12–20 % of cell dry weight (cdw) medium chain length polyhydroxyalkanoates (mcl-PHAs) using different carbon substrates. On the basis of partial 16S rDNA sequence analysis, this bacterium was first identified as Pseudomonas putida and designated as P. putida strain MO2. However, the full 16S rDNA gene sequence from a whole genome sequence analysis of this strain showed 100 % identity to 16S rDNA of Pseudomonas monteilii SB3101 and Pseudomonas monteilii SB3078. The comparison of the cpn60 gene sequence of Pseudomonas sp. strain MO2 with strains of P. putida, P. aeruginosa, P. fluorescens, P. stutzeri, P. syringae, and P. monteilii indicated that this strain is more closely related to P. monteilii than P. putida type strain KT2440. Based on gene sequence similarity index and phylogenetic analyses, some P. putida strains, which were earlier classified as P. putida, are also more closely related to the P. monteilii cluster. Our analyses show that P. putida is a very diverse group with divergent strains, and many strains of P. putida that cluster with P. monteilii species may need to be reclassified.
Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is... more Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal int...
We describe the draft genome sequence of Pseudomonas putida strain LS46, a novel isolate that syn... more We describe the draft genome sequence of Pseudomonas putida strain LS46, a novel isolate that synthesizes medium-chain-length polyhydroxyalkanoates. The draft genome of P. putida LS46 consists of approximately 5.86 million bp, with a G+C content of 61.69%. A total of 5,316 annotated genes and 5,219 coding sequences (CDS) were identified.
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Papers by David Levin