Ectoine Biosynthesis Genes Research Articles

Growth pattern, Protein quantification, and Antibiotic responses of four Halophilic Bacterial Strains in different salt concentration

Abstract: Background: Halophilic bacteria are studied extensively to explore molecular basis of adaptation, enzymatic potential, drug resistance and their other biotechnological implications. Their ability to survive in hypersaline condition is often linked to the potential of biosynthesizing a well-known compatible solute, namely ectoine. Aim: In this study, four bacterial strains, which were previously identified from hypersaline environment were studied for their growth pattern and ectoine biosynthesis proteins expression under varying NaCl concentration. In addition, antibiotic susceptibility of the strains was also investigated. Methods: The growth pattern of the bacterial strains were studied in LB broth supplemented with different NaCl concentration. Bradford method was employed for protein estimation, and optical densities of the samples were quantified through UV-Vis spectrophotometer. Moreover, expression of the ectoine proteins were detected through SDS-PAGE, and disc diffusion method was used for profiling the antibiotic resistance of the strains. Results: The results indicated distinct growth patterns, with each strain demonstrating preferences for specific salinity levels. Notably, Staphylococcus cohnii strain F exhibited optimal growth at 10% NaCl, while Jeotgalicoccus huakuii strain 3.7 thrived at 15% NaCl. Total protein analysis revealed a consistent trend of decreasing protein content with rising NaCl concentrations, elucidating a potential adaptive strategy. Staphylococcus cohnii strain F stood out, maintaining higher protein levels at 20% NaCl, indicative of unique adaptive mechanisms. Antibiotic susceptibility testing uncovered diverse resistance profiles, emphasizing the strain-specific nature of responses. The expression of ectoine biosynthesis genes in Staphylococcus cohnii strain F underlines its adaptive advantage in high salinity. Conclusion: The findings contribute not only to basic microbial physiology knowledge but also hold implications for biotechnological applications and clinical practices, emphasizing the importance of tailored approaches in antibiotic therapy and highlighting the strain-specific nature of bacterial adaptations.

P.585 Differential effects of psychotropic drugs on microbiome composition

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a natural compound, which serves as a protective substance in many halophilic actinobacterial cells. Ectoine biosynthesis genes (ectA, B and C) were PCR amplified from the genomic DNA of deep sea actinobacteria, Nocardiopsis alba NIOT-DSB14. The amplified genes were cloned and nucleotide sequences were determined. Sequencing results revealed that ectA, B and C genes contains 471 bp, 1266 bp, 378 bp long ORF encoding 156, 421 and 125 amino acids, respectively. In silico sequence and phylogenetic analysis of nucleotides and amino acids revealed that the ectA, B and C sequences of Nocardiopsis alba NIOT-DSB14 were conserved in many eubacteria.

Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine.

To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus, a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae, was found to repress ect expression in V. parahaemolyticus In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR, while AphA is an indirect activator of cosR Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness.IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR, which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.

Functional characterization of a major compatible solute in Deep Sea halophilic eubacteria of active volcanic Barren Island, Andaman and Nicobar Islands, India

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a compatible solute, serves as a protective compound in many halophilic eubacterial cells under stress. In this study, the ectoine biosynthesis genes (ectA, B and C) from the genomic DNA of a deep sea eubacteria, Bacillus clausii NIOT-DSB04 was PCR amplified, cloned into the expression vector pQE30 with a 6 × histidine tag and expressed in M15 cells. The lysates of induced cells with diaminobutyric acid aminotransferase and ectoine synthase disclosed two clear expressed bands with molecular masses of 46 kDa and 15 kDa as estimated by SDS-PAGE. The recombinant ectoine synthase activity of the expressed cells was at higher level than that of uninduced cells. In silico sequence and phylogenetic analysis of nucleotides and amino acids revealed that the ectA, B and C sequences of Bacillus clausii NIOT-DSB04 were conserved in many eubacteria.

Molecular characterization and in silico analysis of ectoine biosynthesis genes from the deep sea halophilic eubacteria, Bacillus clausii NIOT-DSB04

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a natural compound, which serves as a protective substance in many halophilic bacterial cells. Ectoine biosynthesis genes (ectA, B and C) were PCR amplified from the genomic DNA of deep sea eubacteria, Bacillus clausii NIOT-DSB04. The amplified genes were cloned and nucleotide sequences were determined. Sequencing results revealed that ectA, B and C genes contains 537 bp, 1281 bp, 405 bp long ORF encoding 178, 426 and 134 amino acids, respectively. In silico sequence and phylogenetic analysis of nucleotides and amino acids revealed that the ectA, B and C sequences of Bacillus clausii NIOT-DSB04 were conserved in many eubacteria.

Ectoine and 5-hydroxyectoine accumulation in the halophile Virgibacillus halodenitrificans PDB-F2 in response to salt stress.

The moderately halophilic bacterium Virgibacillus halodenitrificans PDB-F2 copes with salinity by synthesizing or taking up compatible solutes. The main compatible solutes in this strain were ectoine and hydroxyectoine, as determined by (1)H nuclear magnetic resonance spectroscopy ((1)H-NMR). A high-performance liquid chromatography (HPLC) analysis showed that ectoine was the major solute that was synthesized in response to elevated salinity, while hydroxyectoine was a minor solute. However, the hydroxyectoine/ectoine ratio increased from 0.04 at 3% NaCl to 0.45 at 15% NaCl in the late exponential growth phase. A cluster of ectoine biosynthesis genes was identified, including three genes in the order of ectA, ectB, and ectC. The hydroxyectoine biosynthesis gene ectD was not part of the ectABC gene cluster. Reverse transcription-quantitative polymerase chain reactions (RT-qPCR) showed that the expression of the ect genes was salinity dependent. The expression of ectABC reached a maximum at 12% NaCl, while ectD expression increased up to 15% NaCl. Ectoine and hydroxyectoine production was growth phase dependent. The hydroxyectoine/ectoine ratio increased from 0.018 in the early exponential phase to 0.11 in the stationary phase at 5% NaCl. Hydroxyectoine biosynthesis started much later than ectoine biosynthesis after osmotic shock, and the temporal expression of the ect genes differed under these conditions, with the ectABC genes being expressed first, followed by ectD gene. Increased culture salinity triggered ectoine or hydroxyectoine uptake when they were added to the medium. Hydroxyectoinewas accumulated preferentially when both ectoine and hydroxyectoine were provided exogenously.

Identification and characterization of ectoine biosynthesis genes and heterologous expression of the ectABC gene cluster from Halomonas sp. QHL1, a moderately halophilic bacterium isolated from Qinghai Lake

The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ(70)-dependent promoter and a δ(38)-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain.

Construction and characterization of Methylomicrobium alcaliphilum 20Z knockout mutants defective in sucrose and ectoine biosynthesis genes

Construction and characterization of Methylomicrobium alcaliphilum 20Z knockout mutants defective in sucrose and ectoine biosynthesis genes

Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica

In the halophilic aerobic methylotrophic bacterium Methylophaga thalassica, the genes encoding the enzymes for biosynthesis of the osmoprotectant ectoine were shown to be located in operon ectABC-ask. Transcription of the ect-operon was started from the two promoters homologous to the σ(70)-dependent promoter of Escherichia coli and regulated by protein EctR, whose encoding gene, ectR, is transcribed from three promoters. Genes homologous to ectR of methylotrophs were found in clusters of ectoine biosynthesis genes in some non-methylotrophic halophilic bacteria. EctR proteins of methylotrophic and heterotrophic halophiles belong to the MarR-family of transcriptional regulators but form a separate branch on the phylogenetic tree of the MarR proteins.

Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria

The genes of ectoine biosynthesis pathway were identified in six species of aerobic, slightly halophilic bacteria utilizing methane, methanol or methylamine. Two types of ectoine gene cluster organization were revealed in the methylotrophs. The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA), DABA aminotransferase (EctB) and ectoine synthase (EctC) was found in methanotrophs Methylobacter marinus 7C and Methylomicrobium kenyense AMO1(T). In methanotroph Methylomicrobium alcaliphilum ML1, methanol-utilizers Methylophaga thalassica 33146(T) , Methylophaga alcalica M8 and methylamine-utilizer Methylarcula marina h1(T), the genes forming the ectABC-ask operon are preceded by ectR, encoding a putative transcriptional regulatory protein EctR. Phylogenetic relationships of the Ect proteins do not correlate with phylogenetic affiliation of the strains, thus implying that the ability of methylotrophs to produce ectoine is most likely the result of a horizontal transfer event.

Functional characterization, secondary structure prediction and analysis of ectoine biosynthesis genes from Bacillus halodurans: an osmolyte involved in stress tolerance

An open reading frame encoding the ectoine biosynthesis genes was cloned from the Bacillus halodurans genome. An expression plasmid containing the operon was introduced into Escherichia coli cells, and the recombinant ectoine was quantified. The secondary structure of ectoine biosynthesis proteins were predicted and was quite similar to that of reported proteins from eubacteria.

EctR—A novel transcriptional regulator of ectoine biosynthesis genes in the haloalcaliphilic methylotrophic bacterium Methylophaga alcalica

EctR—A novel transcriptional regulator of ectoine biosynthesis genes in the haloalcaliphilic methylotrophic bacterium Methylophaga alcalica

Heterologous ectoine production in Escherichia coli: By-passing the metabolic bottle-neck

Transcription of the ectoine biosynthesis genes ectA, ectB and ectC from Marinococcus halophilus in recombinant Escherichia coli DH5α is probably initiated from three individual σ70/σA-dependent promoter sequences, upstream of each gene. Consequently, mRNA-fragments containing the single genes and combinations of the genes ectA and ectB or ectB and ectC, respectively, could be detected by Northern blot analysis. Under the control of its own regulatory promoter region (ectUp) a seemingly osmoregulated ectoine production was observed. In addition, aspartate kinases were identified as the main limiting factor for ectoine production in recombinant E. coli DH5α. Co-expression of the ectoine biosynthesis genes and of the gene of the feedback-resistant aspartate kinase from Corynebacterium glutamicum MH20-22B (lysC) led to markedly increased production of ectoine in E. coli DH5α, resulting in cytoplasmic ectoine concentrations comparable to those reached via ectoine accumulation from the medium.