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Functional Analysis of a Glutamine Biosynthesis Protein from a Psychrotrophic Bacterium, Cryobacterium soli GCJ02.

ABSTRACT: A putative glutamine synthetase (GS) was detected in a psychrophilic bacterium, Cryobacterium soli GCJ02. For gaining greater insight into its functioning, the gene was cloned and expressed in a heterologous host, Escherichia coli. The monomer enzyme with a molecular weight of 53.03 kDa was expressed primarily in cytosolic compartment. The enzyme activity was detected using glutamate and ATP. The optimum conditions of its biosynthesis were observed to be 60 °C and pH value 7.5. Its thermostability was relatively high with a half-life of 50 min at 40 °C. GS activity was enhanced in the presence of metal ions such as Mg²⁺ and Mn²⁺, whereas Fe²⁺, Cu²⁺ and Ca²⁺ proved inhibitory. The consensus pattern [EXE]-D-KP-[XGXGXH] in the GS lies between residues 132 and 272. The catalytic active sites consisting of EAE and NGSGMH were verified by site-directed mutagenesis. Based on the analysis of the consensus pattern, the GS/glutamate synthase cycle of C. soli GCJ02 is expected to contribute to the GS synthesic activity.

SUBMITTER: Gong C

PROVIDER: S-EPMC7105534 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We show for soil bacterium Enterobacter soli LF7 that the possession of an indole-3-acetic acid catabolic (iac) gene cluster is causatively linked to the ability to utilize the plant hormone indole-3-acetic acid (IAA) as a carbon and energy source. Genome-wide transcriptional profiling by mRNA sequencing revealed that these iac genes, chromosomally arranged as iacHABICDEFG and coding for the transformation of IAA to catechol, were the most highly induced (>29-fold) among the relatively few (<1%) differentially expressed genes in response to IAA. Also highly induced and immediately downstream of the iac cluster were genes for a major facilitator superfamily protein (mfs) and enzymes of the ?-ketoadipate pathway (pcaIJD-catBCA), which channels catechol into central metabolism. This entire iacHABICDEFG-mfs-pcaIJD-catBCA gene set was constitutively expressed in an iacR deletion mutant, confirming the role of iacR, annotated as coding for a MarR-type regulator and located upstream of iacH, as a repressor of iac gene expression. In E. soli LF7 carrying the DNA region upstream of iacH fused to a promoterless gfp gene, green fluorescence accumulated in response to IAA at concentrations as low as 1.6 ?M. The iacH promoter region also responded to chlorinated IAA, but not other aromatics tested, indicating a narrow substrate specificity. In an iacR deletion mutant, gfp expression from the iacH promoter region was constitutive, consistent with the predicted role of iacR as a repressor. A deletion analysis revealed putative -35/-10 promoter sequences upstream of iacH, as well as a possible binding site for the IacR repressor.IMPORTANCE Bacterial iac genes code for the enzymatic conversion of the plant hormone indole-3-acetic acid (IAA) to catechol. Here, we demonstrate that the iac genes of soil bacterium Enterobacter soli LF7 enable growth on IAA by coarrangement and coexpression with a set of pca and cat genes that code for complete conversion of catechol to central metabolites. This work contributes in a number of novel and significant ways to our understanding of iac gene biology in bacteria from (non-)plant environments. More specifically, we show that LF7's response to IAA involves derepression of the MarR-type transcriptional regulator IacR, which is quite fast (less than 25 min upon IAA exposure), highly specific (only in response to IAA and chlorinated IAA, and with few genes other than iac, cat, and pca induced), relatively sensitive (low micromolar range), and seemingly tailored to exploit IAA as a source of carbon and energy.

Dataset Information

Functional Analysis of a Glutamine Biosynthesis Protein from a Psychrotrophic Bacterium, Cryobacterium soli GCJ02.

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