Development of a genetic transformation system for an alga-lysing bacterium.
ABSTRACT: Four marine bacteria, Alteromonas sp. strains A27, A28, A29, and A30, that lyse the diatom Skeletonema costatum NIES-324 were isolated from coastal seawater samples. They were also able to lyse the diatoms Thalassiosira sp. and Eucampia zodiacs and the raphidophycean flagellate Chattonella antiqua. Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in Alteromonas sp. strains A28 and A29, respectively. These plasmids appeared to be similar based on size and restriction site analysis. A shuttle vector that replicates in Escherichia coli and Alteromonas sp. strain A28 was constructed by fusing pAS28 and E. coli vector pCRIIc. The 16-kbp chimeric plasmid, designated pASS1, had the ability to transform strain A28 at a frequency of 10(6) transformants per microg of DNA. Deletion analysis of pASS1 showed that the 4.7-kb EcoRI-HindIII region of pAS28 was essential for plasmid maintenance in strain A28. This EcoRI-HindIII fragment contained an open reading frame which appeared to encode a 708-amino-acid protein.
Project description:Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents. The natural substrates for these enzymes are unknown. The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate. A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressing this activity was found in a 1.74-kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues. Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.
Project description:Here, we report the draft genome sequences of Bacillus subtilis A1, Sphingobacterium sp. strain A3, and Pseudomonas sp. strain A29; Sphingobacterium sp. A3 and Pseudomonas sp. A29 were identified as Bacillus velezensis strain A3 and Bacillus subtilis strain A29, respectively, after a quality control check of the whole-genome sequences deposited in the NCBI database. These bacteria exhibit tremendous production of siderophores and significant antimicrobial potential. When inoculated on maize, these isolates increase its yield.
Project description:The gene encoding an extracellular chitinase from marine Alteromonas sp. strain O-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase of Alteromonas sp. strain O-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.
Project description:In two previous studies dealing with lactic acid bacteria (LAB) from modified-atmosphere-packaged (MAP) broiler products and a broiler processing plant, several isolates remained unidentified. According to 16S rRNA gene sequence analysis, 36 isolates were assigned to the genus Enterococcus. Numerical analysis of combined HindIII and EcoRI ribopatterns of these isolates resulted in species-specific clusters that were congruent with the clusters obtained by both DNA-directed RNA polymerase subunit A (rpoA) and phenylalanyl-tRNA synthetase ? chain (pheS) housekeeping gene analyses. In the analyses, a group of five isolates distinct from any known enterococcal species clustered together. The five isolates were positioned in the Enterococcus avium group, with E. devriesei being the closest phylogenetic neighbor. The DNA-DNA hybridization levels with E. devriesei ranged from 28.8 to 54.3% and indicated that these strains represented a novel species. The name Enterococcus viikkiensis sp. nov. is proposed, with strain DSM 24043(T) (LMG 26075(T)) being the type strain. Our study demonstrated that the identification of enterococci within the E. avium phylogenetic group demands polyphasic taxonomic approaches. The rpoA and pheS gene similarities (99.0 to 99.2% and 94.3 to 95.4%, respectively) between E. viikkiensis and its closest phylogenetic neighbor, E. devriesei, were higher than those previously reported within the enterococci. In addition, the phenotypic profiles of the species in the E. avium group were also highly similar, and some traits were found to be misleading for enterococci, such as E. viikkiensis does not grow at 45°C. The numerical analysis of combined HindIII and EcoRI ribopatterns was of considerable assistance in distinguishing enterococcal species within the E. avium group.
Project description:Upon UV irradiation, Bacillus subtilis spore DNA accumulates the novel thymine dimer 5-thyminyl-5,6-dihydrothymine. Spores can repair this "spore photoproduct" (SP) upon germination either by the uvr-mediated general excision repair pathway or by the SP-specific spl pathway, which involves in situ monomerization of SP to two thymines by an enzyme named SP lyase. Mutants lacking both repair pathways produce spores that are extremely sensitive to UV. For cloning DNA that can repair a mutation in the spl pathway called spl-1, a library of EcoRI fragments of chromosomal DNA from B. subtilis 168 was constructed in integrative plasmid pJH101 and introduced by transformation into a mutant B. subtilis strain that carries both the uvrA42 and spl-1 mutations, and transformants whose spores exhibited UV resistance were selected by UV irradiation. With a combination of genetic and physical mapping techniques, the DNA responsible for the restoration of UV resistance was shown to be present on a 2.3-kb EcoRI-HindIII fragment that was mapped to a new locus in the metC-pyrD region of the B. subtilis chromosome immediately downstream from the pstI gene. The spl coding sequence was localized on the cloned fragment by analysis of in vitro-generated deletions and by nucleotide sequencing. The spl nucleotide sequence contains an open reading frame capable of encoding a 40-kDa polypeptide that shows regional amino acid sequence homology to DNA photolyases from a number of bacteria and fungi.
Project description:Studying the physiology and genomics of cultured hydrolytic bacteria is a valuable approach to decipher the biogeochemical cycling of marine polysaccharides, major nutrients derived from phytoplankton and macroalgae. We herein describe the profound potential of Alteromonas sp. 76-1, isolated from alginate-enriched seawater at the Patagonian continental shelf, to degrade the algal polysaccharides alginate and ulvan. Phylogenetic analyses indicated that strain 76-1 might represent a novel species, distinguished from its closest relative (Alteromonas naphthalenivorans) by adaptations to their contrasting habitats (productive open ocean vs. coastal sediments). Ecological distinction of 76-1 was particularly manifested in the abundance of carbohydrate-active enzymes (CAZymes), consistent with its isolation from alginate-enriched seawater and elevated abundance of a related OTU in the original microcosm. Strain 76-1 encodes multiple alginate lyases from families PL6, PL7, PL17, and PL18 largely contained in two polysaccharide utilization loci (PUL), which may facilitate the utilization of different alginate structures in nature. Notably, ulvan degradation relates to a 126 Kb plasmid dedicated to polysaccharide utilization, encoding several PL24 and PL25 ulvan lyases and monomer-processing genes. This extensive and versatile CAZyme repertoire allowed substantial growth on polysaccharides, showing comparable doubling times with alginate (2 h) and ulvan (3 h) in relation to glucose (3 h). The finding of homologous ulvanolytic systems in distantly related Alteromonas spp. suggests CAZyme plasmids as effective vehicles for PUL transfer that mediate niche gain. Overall, the demonstrated CAZyme repertoire substantiates the role of Alteromonas in marine polysaccharide degradation and how PUL exchange influences the ecophysiology of this ubiquitous marine taxon.
Project description:Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to alpha-chitin and beta-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35 degrees C, respectively, and even at 10 degrees C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.
Project description:A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group.
Project description:Alteromonas spp. are Gram-negative, aerobic, marine bacteria. Here, we report the draft genome sequence of Alteromonas sp. strain KS69, isolated from Narragansett Bay deep water samples. Unpublished preliminary data suggest that KS69 reduces expression of the 3-oxo-C12-HSL-dependent, virulence-associated gene lasB of Pseudomonas aeruginosa PAO1, suggesting that it produces a quorum sensing inhibitor.
Project description:Enzymes (endonucleases) are coupled to constitutional dynamic networks to stimulate the selection of a constituent and cascaded emergence of a new network. This is exemplified with the EcoRI-dictated depletion of a network and selection of a constituent that activates the cascaded emergence of a new network. The new network is further depleted by HindIII to a selected constituent that can be coupled to the cascaded emergence of a dynamic network. In addition, upon subjecting a [3 × 3] constitutional dynamic network to endonucleases EcoRI and HindIII, the programmed hierarchical selection of [2 × 2] constitutional dynamic networks followed by the biocatalytic selection of a constituent for the subsequent emergence of new networks is demonstrated.