Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases.
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ABSTRACT:
SUBMITTER: Chavagnat F
PROVIDER: S-EPMC1217806 | biostudies-other | 1996 Oct
REPOSITORIES: biostudies-other
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Project description:Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II' (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.
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Project description:The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.
| S-EPMC1148705 | biostudies-other
Project description:Mucoid strains of Pseudomonas aeruginosa produce a viscous exopolysaccharide called alginate and also express alginate lyase activity which can degrade this polymer. By transposon mutagenesis and gene replacement techniques, the algL gene encoding a P. aeruginosa alginate lyase enzyme was found to reside between algG and algA within the alginate biosynthetic gene cluster at 35 min on the P. aeruginosa chromosome. DNA sequencing data for algL predicted a protein product of ca. 41 kDa, including a 27-amino-acid signal sequence, which would be consistent with its possible localization in the periplasmic space. Expression of the algL gene in Escherichia coli cells resulted in the expression of alginate lyase activity and the appearance of a new protein of ca. 39 kDa detected on sodium dodecyl sulfate-polyacrylamide gels. In mucoid P. aeruginosa strains, expression of algL was regulated by AlgB, which also controls expression of other genes within the alginate gene cluster. Since alginate lyase activity is associated with the ability to produce and secrete alginate polymers, alginate lyase may play a role in alginate production.
| S-EPMC204930 | biostudies-other
Project description:Alginate is a linear polysaccharide produced mainly by brown algae in marine environments. Alginate consists of a linear block copolymer made up of two monomeric units, ?-d-mannuronate (M) and its C-5 epimer ?-l-guluronate (G). Alginate lyases are polysaccharide lyases (PL) that degrade alginate via a ?-elimination reaction. These enzymes play an important role in marine carbon recycling and also have widespread industrial applications. So far, more than 1,774 alginate lyase sequences have been identified and are distributed into 7 PL families. In this review, the folds, conformational changes during catalysis, and catalytic mechanisms of alginate lyases are described. Thus far, structures for 15 alginate lyases have been solved and are divided into 3 fold classes: the ?-jelly roll class (PL7, -14, and -18), the (?/?)n toroid class (PL5, -15, and -17), and the ?-helix fold (PL6). These enzymes adopt two different mechanisms for catalysis, and three kinds of conformational changes occur during this process. Moreover, common features in the structures, conformational changes, and catalytic mechanisms are summarized, providing a comprehensive understanding on alginate lyases.
| S-EPMC5772247 | biostudies-literature
Project description:Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer of key interest for human health. To increase knowledge on alginate degradation in the gut, a gene catalog from human feces was mined for potential alginate lyases (ALs). The predicted ALs were present in nine species of the Bacteroidetes phylum, of which two required supplementation of an endo-acting AL, expected to mimic cross-feeding in the gut. However, only a new isolate grew on alginate. Whole-genome sequencing of this alginate-utilizing isolate suggested that it is a new Bacteroides ovatus strain harboring a polysaccharide utilization locus (PUL) containing three ALs of families: PL6, PL17, and PL38. The BoPL6 degraded polyG to oligosaccharides of DP 1-3, and BoPL17 released 4,5-unsaturated monouronate from polyM. BoPL38 degraded both alginates, polyM, polyG, and polyMG, in endo-mode; hence, it was assumed to deliver oligosaccharide substrates for BoPL6 and BoPL17, corresponding well with synergistic action on alginate. BoPL17 and BoPL38 crystal structures, determined at 1.61 and 2.11 Å, respectively, showed (α/α)6-barrel + anti-parallel β-sheet and (α/α)7-barrel folds, distinctive for these PL families. BoPL17 had a more open active site than the two homologous structures. BoPL38 was very similar to the structure of an uncharacterized PL38, albeit with a different triad of residues possibly interacting with substrate in the presumed active site tunnel. Altogether, the study provides unique functional and structural insights into alginate-degrading lyases of a PUL in a human gut bacterium.IMPORTANCEHuman ingestion of sustainable biopolymers calls for insight into their utilization in our gut. Seaweed is one such resource with alginate, a major cell wall component, used as a food hydrocolloid and for encapsulation of pharmaceuticals and probiotics. Knowledge is sparse on the molecular basis for alginate utilization in the gut. We identified a new Bacteroides ovatus strain from human feces that grew on alginate and encoded three alginate lyases in a gene cluster. BoPL6 and BoPL17 show complementary specificity toward guluronate (G) and mannuronate (M) residues, releasing unsaturated oligosaccharides and monouronic acids. BoPL38 produces oligosaccharides degraded by BoPL6 and BoPL17 from both alginates, G-, M-, and MG-substrates. Enzymatic and structural characterization discloses the mode of action and synergistic degradation of alginate by these alginate lyases. Other bacteria were cross-feeding on alginate oligosaccharides produced by an endo-acting alginate lyase. Hence, there is an interdependent community in our guts that can utilize alginate.
| S-EPMC10617595 | biostudies-literature
Project description:Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (kcat) for alginate of 0.60 ± 0.02 s(-1), 3.7 ± 0.3 s(-1), 4.5 ± 0.5 s(-1), and 7.1 ± 0.2 s(-1), respectively. The Km values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.
| S-EPMC4325137 | biostudies-literature
Project description:Alginates are polysaccharides composed of 1-4-linked beta-D-mannuronic acid and alpha-L-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a beta-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca(2+). All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.
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Project description:BackgroundThe Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure.ResultsThe reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3' non-coding region) positioned 19-bp away from the lacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing.ConclusionThe ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of the E. coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications.
| S-EPMC7296898 | biostudies-literature
Project description:Using an oligonucleotide hybridization probe, we have mapped the structural gene for the delta subunit of Escherichia coli DNA polymerase III holoenzyme to 14.6 centisomes of the chromosome. This gene, designated holA, was cloned and sequenced. The sequence of holA matches precisely four amino acid sequences obtained for the amino terminus of delta and three internal tryptic peptides. A holA-overproducing plasmid that directs the expression of delta up to 4% of the soluble protein was constructed. Sequence analysis of holA revealed a 1,029-bp open reading frame that encodes a protein with a predicted molecular mass of 38,703 Da. holA may reside downstream of rlpB in an operon, perhaps representing yet another link between structural genes for the DNA polymerase III holoenzyme and proteins involved in membrane biogenesis. These and other features are discussed in terms of genetic regulation of delta-subunit synthesis.
| S-EPMC207382 | biostudies-other
Project description:Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZ?A-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.
| S-EPMC5811067 | biostudies-literature