Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: The multi-functional DHAP shunt of E. coli . ( A ) Genomic organization of the DHAP shunt operon and neighboring genes in E. coli ATCC 25922. The operon specifically consists of mtnK , mtnA , ald2 , and an annotated permease of unknown function . The pfs gene, conserved across all E. coli , is encoded in a different region of the E. coli genome. ( B ) Multiple sequence alignment of the DHAP shunt 5′-UTR region from 1,569 E. coli genomes containing the DHAP shunt operon. Positions matching the ATCC 25922 reference are shown in gray, while mismatches are shown in red. ( C and D ) Biochemical reactions catalyzed by the DHAP shunt pathway enzymes, with 5′-deoxyadenosine ( C ) or 5′-methylthioadenosine ( D ) as initial substrate.

Article Snippet: Transcriptomic reads were mapped to ATCC 25922 reference sequences NZ_CP032085.1 (chromosome), NZ_CP032087.1 , NZ_CP032088.1 , and NZ_CP032086.1 (plasmids).

Techniques: Functional Assay, Sequencing

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: Regulation of E. coli ATCC 25922 transcriptome during growth on glucose, 5dR, or carbon starvation. Volcano plots of changes in ATCC 25922 transcript abundance for ( A ) wild-type ATCC 25922 grown with 5dR versus glucose, ( B ) MtnR deletion strain (∆ mtnR ) versus wild-type ATCC 25922 grown with glucose, and ( C ) ATCC 25922 incubated under carbon starvation conditions versus carbon replete (glucose) conditions. Gray lines indicate 2.5 log 2 -fold change thresholds; DHAP shunt genes are highlighted in pink. ( A ) DHAP shunt genes are strongly upregulated (~7.5 log 2 ) in the presence of 5dR. ( B ) Deletion of MtnR (∆ mtnR ) results in ~5 log 2 increased expression of DHAP shunt genes compared to the wild-type strain in the presence of glucose. ( C ) Carbon starvation only moderately upregulated DHAP shunt gene expression. ( D ) Inventory of genes differentially expressed under 5dR growth and carbon starvation relative to growth on glucose. Carbon starvation triggers a large change in the proteome compared to growth on 5dR, indicating the proteome change during growth on 5dR is specific to the substrate and not a general stress response.

Article Snippet: Transcriptomic reads were mapped to ATCC 25922 reference sequences NZ_CP032085.1 (chromosome), NZ_CP032087.1 , NZ_CP032088.1 , and NZ_CP032086.1 (plasmids).

Techniques: Incubation, Expressing, Gene Expression

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: DHAP shunt 5′-UTR regions required for operon expression and repression by MtnR. ( A ) E. coli ATCC 25922 DHAP shunt genomic context and sections of 5′-UTR used for construction of plasmids (pLacZ###) with promoter– lacZ fusions. ( B, D ) LacZ activity assays from cell extracts of wild-type E. coli ATCC 25922 and ( C, E ) LacZ activity assays from cell extracts of MtnR deletion strain (Δ mtnR ) containing the indicated DHAP shunt 5′-UTR– lacZ fusion plasmids when grown with either 5 mM glucose (Glc), 5 mM glucose plus 5 mM 5dR (Glc + 5dR), or 5dR as the carbon source. The pLacZ control is a Lac promoter– lacZ fusion without added IPTG. Average and standard deviation error bars are for n = 3 independent replicates. ** P > 0.05, * P > 0.1; two-tailed t -test. ( F ) Log 2 -fold change in mtnK mRNA expression from transcriptomic analysis ( ; ) and measured by qRT-PCR for wild-type ATCC 25922 (WT), mtnR deletion strain (Δ mtnR ), and mtnR deletion strain complemented with mtnR from a plasmid (Δ mtnR + pmtnR). Tetracycline (10 ng/mL) was added to induce expression of mtnR from pmtnR plasmid. qRT-PCR average and standard deviation error bars are for n = 4 independent biological replicates except for WT Glc + 5dR versus Glc ( n = 8) and Δ mtnR + pmtnR Glc + 5dR versus Glc ( n = 3). ** P > 0.05; two-tailed t -test.

Article Snippet: Transcriptomic reads were mapped to ATCC 25922 reference sequences NZ_CP032085.1 (chromosome), NZ_CP032087.1 , NZ_CP032088.1 , and NZ_CP032086.1 (plasmids).

Techniques: Expressing, Activity Assay, Control, Standard Deviation, Two Tailed Test, Quantitative RT-PCR, Plasmid Preparation

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: DHAP shunt expression is not activated by other alternative growth substrates. LacZ activity assays from cell extracts of E. coli ATCC 25922 containing the DHAP shunt 5′-UTR– lacZ fusion plasmid placZ255 and grown with either 5 mM glucose, NANA, l -fucose, l -arabinose, l -rhamnose, or 5-deoxy- d -ribose (5dR) as the sole carbon source. Average and standard deviation error bars are for n = 3 independent replicates. ** P > 0.05.

Article Snippet: Transcriptomic reads were mapped to ATCC 25922 reference sequences NZ_CP032085.1 (chromosome), NZ_CP032087.1 , NZ_CP032088.1 , and NZ_CP032086.1 (plasmids).

Techniques: Expressing, Activity Assay, Plasmid Preparation, Standard Deviation

Journal: Journal of Bacteriology

Article Title: Escherichia coli is poised to grow using 5′-deoxynucleosides via MtnR and CRP regulation of DHAP shunt gene expression

doi: 10.1128/jb.00280-25

Figure Lengend Snippet: CRP is required for growth of E. coli ATCC 25922 using 5dR as a substrate. ( A and B ) Growth of wild-type ATCC 25922 (diamonds), CRP deletion strain (Δ crp ; triangles), and CRP deletion strain complemented with crp expressed from a plasmid (Δ crp + pCRP; circles) with ( A ) 5dR or ( B ) glucose as the sole carbon source. Average and standard deviation error bars are for n = 4 for growth on 5dR and n = 5 for growth on glucose. ( C ) Diauxic growth observed when wild-type ATCC 25922 is grown on a combination of 1 mM glucose plus 8 mM 5dR (diamonds), as compared to cultures grown with 1 mM glucose (triangles) or 8 mM 5dR (circles) as the sole carbon source. Dotted lines and fit parameters are for exponential regression fit to the growth data. Average and standard deviation error bars are for n = 5 independent biological replicates.

Article Snippet: Transcriptomic reads were mapped to ATCC 25922 reference sequences NZ_CP032085.1 (chromosome), NZ_CP032087.1 , NZ_CP032088.1 , and NZ_CP032086.1 (plasmids).

Techniques: Plasmid Preparation, Standard Deviation

Journal: bioRxiv

Article Title: S-SELeCT: A Human-Evolved Serine Integrase System for Efficient Large-Cargo Genome Integration

doi: 10.64898/2026.01.30.702954

Figure Lengend Snippet: Minichromosome assay ( A ) Site A-splice acceptor-3’ GFP-polyA was cloned in a plasmid with 2.5 - 5 kb Site A genomic sequence on each side, along with other elements needed for EBNA1-mediated extrachromosomal maintenance in human cells (EBNA1 expression, oriP, puromycin-resistance marker). The 20 kb (SAR MAX, not shown) and 15 kb (SAR 1) plasmids have 5 kb and 2.5 kb genomic Site A sequence on each side (10 and 5 kb total), respectively. ( B ) When the mini-chromosome assay was used in screens, we used the depicted alternative-splicing expression construct to produce the needed solo and fused forms of each variant integrase. ( C ) Results from individual tests of the top-performing variants in the mini-chromosome assay. Assay was performed for 72 hours in HEK293 cells with the Site A SAR1 mini-chromosome that stably expressed the indicated variant using an alternative-splicing cassette from the H11 locus. N=3 biological replicates, error bars are standard error (STDEV/SQRT).

Article Snippet: Cultures of cell lines with the SAR1 or SARMAX mini-chromosome were additionally supplemented with 1-2 ug/mL puromycin (InvivoGen ant-pr-1).

Techniques: Clone Assay, Plasmid Preparation, Sequencing, Expressing, Marker, Alternative Splicing, Construct, Variant Assay, Stable Transfection

Journal: bioRxiv

Article Title: S-SELeCT: A Human-Evolved Serine Integrase System for Efficient Large-Cargo Genome Integration

doi: 10.64898/2026.01.30.702954

Figure Lengend Snippet: Transient variant expression, mini-chromosome assay ( A ) Drawing of the plasmid used to express solo and fused forms of variants via alternative-splicing in transient-delivery assays. hU6-driven expression cassettes for guide RNAs g10 and g6 were also present in these vectors (not shown). ( B ) Results from V12 (ratios E, F, G) and V30 (ratios F, G) expressed via the CAG-promoter in the transient expression mini-chromosome assay. ( C ) Results for mPGK-promoter driven expression of V34 (ratios E, G), V7 (ratios E, F) and V16 (ratio E), normalized to CAG-driven V12 ratio E. All experiments were performed over 72 hours in HEK293 cells that contained the mini-chromosome (Site A SAR1). N=3 biological replicates and error-bars are standard error.

Article Snippet: Cultures of cell lines with the SAR1 or SARMAX mini-chromosome were additionally supplemented with 1-2 ug/mL puromycin (InvivoGen ant-pr-1).

Techniques: Variant Assay, Expressing, Plasmid Preparation, Alternative Splicing