Structure of a Core Promoter in Bifidobacterium longum NCC2705

Abstract

Bacterial promoters consist of core sequence motifs termed –35 and –10 boxes. The consensus motifs are TTGACA and TATAAT, respectively, which were identified from leading investigations on Escherichia coli. However, the consensus sequences are not likely to fit genetically divergent bacteria. The sigma factor of the genus Bifidobacterium has a characteristic polar domain in the N terminus, suggesting the possibility of specific promoter recognition. We reevaluated the structure of Bifidobacterium longum NCC2705 promoters and compared them to other bacteria. Transcriptional start sites (TSSs) of the B. longum NCC2705 strain were identified using transcriptome sequencing (RNA-Seq) analysis to extract promoter regions. Conserved motifs of a bifidobacterial promoter were determined using regions upstream of TSSs and a hidden Markov model. As a result, consensus motifs of the –35 and –10 boxes were TTGTGC and TACAAT, respectively. To assess each base of both motifs, we constructed 37 plasmids based on pKO403-TPCTcon, including the hup promoter connected with a chloramphenicol acetyltransferase as a reporter gene. This reporter assay showed two optimal motifs of the –35 and –10 boxes, namely, TTGNNN and TANNNT, respectively. We further analyzed spacer lengths between the –35 and –10 boxes via a bioinformatics approach. The spacer lengths predominant in bacteria have been generally reported to be approximately 17 bp. In contrast, the predominant spacer lengths in the genus Bifidobacterium and related species were 11 bp, in addition to 17 bp. A reporter assay to assess the spacer lengths indicated that the 11-bp spacer length produced unusually high activity. IMPORTANCE The structures of sigma factors vary among bacterial strains, indicating that recognition rules may also vary. Therefore, we investigated the promoter structure of Bifidobacterium longum NCC2705 using a bioinformatics approach and wet analyses. The most frequent and optimal motifs were similar to other bacterial consensus motifs. The optimal spacer length between the two boxes was reported to be 17 bp. It is widely applied to a bioinformatics approach for other bacteria. Unexpectedly, conserved spacer lengths were 11 bp as well as 17 bp in the genus Bifidobacterium. Moreover, the sigma factor of the genus Bifidobacterium has a characteristic domain in the N terminus which may contribute to the additional functions. Hence, it would be valuable to reevaluate the promoter in other organisms.

Recent Development of Probiotic Bifidobacteria for Treating Human Diseases

“…The promoter core motif sequences were identified in silico using the hidden Markov model on the upstream sequences of the transcriptional start site of various coding genes in Bifidobacterium. This approach was used to identify the various putative constitutive promoter sequences in B. longum NCC2705 ( Kozakai et al, 2020 ; Kozakai et al, 2021 ) and B. longum 105-A ( Sakanaka et al, 2014 ). Additionally, the space between the -35 and -10 sequences can range from 11 to 18 nucleotides in length, where a shorter space length was found to improve the transcription levels ( Kozakai et al, 2020 ).…”

“…This approach was used to identify the various putative constitutive promoter sequences in B. longum NCC2705 ( Kozakai et al, 2020 ; Kozakai et al, 2021 ) and B. longum 105-A ( Sakanaka et al, 2014 ). Additionally, the space between the -35 and -10 sequences can range from 11 to 18 nucleotides in length, where a shorter space length was found to improve the transcription levels ( Kozakai et al, 2020 ).…”

“…Second, the optimal distance between the RBS site and the start codon influences the rate of protein expression ( Fukiya et al, 2018 ). Interestingly, certain studies have shown that the predominant conserved Shine-Dalgarno sequence in Bifidobacterium differs from conventional microbes where the most common of 6-mer consensus RBS in B. longum is AAGGAG as compared to the common AGGAGG ( He et al, 2012b ; Kozakai et al, 2020 ). Additionally, the space between the RBS and the start codon was found to be optimal at 5 nucleotides apart, where fewer than 5 nucleotides apart result in translation repression.…”

Human Milk Oligosaccharide Utilization in Intestinal Bifidobacteria is Governed by a Global Transcriptional Regulator NagR

“…NagR-binding motifs were visualized via WebLogo (64). Positions of the −10 and −35 promoter elements were determined via similar PMW-based searches based on data available for B. breve (65) and B. longum (66). Promoter regions were aligned using Pro-Coffee (67).…”

Phage Annotation Guide: Guidelines for Assembly and High-Quality Annotation

“… 71 We recommend that you consult the literature for the latest consensus sequence for the bacterium of interest. 72 One of the authors (A.M.K.) only records sequences that differ by two nucleotides or less from the consensus.…”