The nodD locus from Azorhizobium caulinodans is flanked by two repetitive elements

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The Azorhizobium caulinodans (Ac) nodD gene is crucial for regulating nodulation in Sesbania rostrata.
• Understanding the genetic elements surrounding nodD can provide insights into its evolutionary history and regulation.
• Previous studies have identified repeated elements near nodD, but their function and evolutionary significance remain unclear.

Purpose of the Study:

• To analyze the DNA sequence surrounding the Azorhizobium caulinodans nodD gene.
• To investigate the role of flanking repeated elements (epsilon 1 and delta 1) in nodD evolution and function.
• To determine the transcriptional regulation of nodD and associated open reading frames (ORF1 and ORF2).

Main Methods:

• DNA sequencing and analysis of the region surrounding the Ac nodD gene.
• Identification and characterization of open reading frames (ORF1, ORF2) and repeated elements (epsilon 1, delta 1).
• Insertion mutagenesis of epsilon 1 and delta 1 to assess their impact on nodulation.
• LacZ fusion studies to investigate nodD promoter activity and transcriptional regulation.

Main Results:

• Two small open reading frames (ORF1 and ORF2) and a repeated element (epsilon 1) were identified upstream of nodD.
• The epsilon 1 element shares similarity with insertion element IS51 from Pseudomonas savastanoi.
• The epsilon 1 and delta 1 elements flank a GC content shift and exist in multiple copies in the Ac genome, suggesting a role in horizontal gene transfer.
• Insertion mutations in epsilon 1 and delta 1 did not affect nodulation operon induction or nodulation behavior on Sesbania rostrata.
• LacZ fusion studies indicated constitutive transcription of nodD, with its promoter overlapping ORF1.
• No promoter activity was detected in the direction of ORF1 and ORF2.
• A nod box-related motif, lacking the ATC-9-bp-GAT palindrome crucial for NodD-mediated stimulation, was found in the nodD-ORF1 intergenic region.

Conclusions:

• The repeated elements epsilon 1 and delta 1 likely facilitated the horizontal transfer of the nodD gene during evolution.
• The nodD gene is constitutively transcribed, and its expression is regulated by a promoter overlapping ORF1.
• The identified nod box-related motif deviates from active motifs, suggesting a modified regulatory mechanism.
• Despite the presence of flanking elements and a modified nod box, nodD function and nodulation appear unaffected in Sesbania rostrata under tested conditions.