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PreprintCited by 9Bacterial Genetics and BiotechnologyBiochemical and Structural CharacterizationBacterial biofilms and quorum sensing

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A role for the ATP-dependent DNA ligase Lig E of Neisseria gonorrhoeae in biofilm formation

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Pan, Jolyn, Hicks, Joanna, Williamson, Adele·2023·BMC Microbiology
DOI 10.21203/rs.3.rs-3396984/v1PMC PMC10799422

Preprint — published as 10.1186/s12866-024-03193-9 · Paper page

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Abstract

The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection characteristics. This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells which coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation, providing insight into the binding dynamics of these minimal DNA ligases.

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Figures

Fig. 1 (A) Schematic of the genetic constructs for His-tagged Ngo-lig E (ngo-Lig E-His), the Ngo-lig E deletion mutant (Δngo-Lig E) and constitutively upregulated Ngo-Lig E (opaB-Ngo-Lig E). (B) Growth of N. gonorrhoeae variants monitored by OD600. (C) Number of viable cells of N. gonorrhoeae variants monitored by CFU counts for each culture. Points are the mean of triplicate measurements and error bars represent the standard error of the mean
Fig. 1 (A) Schematic of the genetic constructs for His-tagged Ngo-lig E (ngo-Lig E-His), the Ngo-lig E deletion mutant (Δngo-Lig E) and constitutively upregulated Ngo-Lig E (opaB-Ngo-Lig E). (B) Growth of N. gonorrhoeae variants monitored by OD600. (C) Number of viable cells of N. gonorrhoeae variants monitored by CFU counts for each culture. Points are the mean of triplicate measurements and error bars represent the standard error of the meanCC BY · PubMed Central
Fig. 2 Comparisons of gene expression with normalisation to the 16S gene. (A) Fold changes of ngo-Lig E-His and opa-ngo-Lig E compared to wild-type N. gonorrhoeae under either planktonic or biofilm conditions. (B) Fold changes of wild type, ngo-Lig E-His, and opa-ngo-Lig E gene expression under biofilm conditions compared to planktonic conditions. Points are the mean of triplicate measurements and error bars represent the standard error of the mean. Significance values are given as * P ≤ 0.05; ** P ≤ 0.01. Comparisons which showed no significant difference (P > 0.05) are not indicated
Fig. 2 Comparisons of gene expression with normalisation to the 16S gene. (A) Fold changes of ngo-Lig E-His and opa-ngo-Lig E compared to wild-type N. gonorrhoeae under either planktonic or biofilm conditions. (B) Fold changes of wild type, ngo-Lig E-His, and opa-ngo-Lig E gene expression under biofilm conditions compared to planktonic conditions. Points are the mean of triplicate measurements and error bars represent the standard error of the mean. Significance values are given as * P ≤ 0.05; ** P ≤ 0.01. Comparisons which showed no significant difference (P > 0.05) are not indicatedCC BY · PubMed Central
Fig. 3 Damage and oxidative stress assays with N. gonorrhoeae strains. (A) Survival after treatment with hydrogen peroxide. (B) Survival after irradiation with UV light. (C) Survival after treatment with nalidixic acid. Points are the mean of triplicate measurements and error bars represent the standard error of the mean. Significance values are given as * P ≤ 0.05. Comparisons which showed no significant difference (P > 0.05) are not indicated
Fig. 3 Damage and oxidative stress assays with N. gonorrhoeae strains. (A) Survival after treatment with hydrogen peroxide. (B) Survival after irradiation with UV light. (C) Survival after treatment with nalidixic acid. Points are the mean of triplicate measurements and error bars represent the standard error of the mean. Significance values are given as * P ≤ 0.05. Comparisons which showed no significant difference (P > 0.05) are not indicatedCC BY · PubMed Central
Fig. 4 (A) Biofilm formation in liquid culture by N. gonorrhoeae strains. (B) Proportion of Ngo-Lig E mutants adhering to epithelial cells. (C) Proportion of Ngo-Lig E mutants remaining in the non-adhered (planktonic) state when co-cultured with epithelial cells (D) Proportion of Ngo-Lig E mutants invading epithelial cells relative to the total N. gonorrhoeae (planktonic and adhered). Points are the mean of triplicate measurements and error bars represent the standard error of the mean. Significance values are given as * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Comparisons which showed no significant difference (P > 0.05) are not indicated
Fig. 4 (A) Biofilm formation in liquid culture by N. gonorrhoeae strains. (B) Proportion of Ngo-Lig E mutants adhering to epithelial cells. (C) Proportion of Ngo-Lig E mutants remaining in the non-adhered (planktonic) state when co-cultured with epithelial cells (D) Proportion of Ngo-Lig E mutants invading epithelial cells relative to the total N. gonorrhoeae (planktonic and adhered). Points are the mean of triplicate measurements and error bars represent the standard error of the mean. Significance values are given as * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Comparisons which showed no significant difference (P > 0.05) are not indicatedCC BY · PubMed Central
Fig. 5 (A) Schematic of FAM-labelled DNA substrates with ligatable breaks, (B) Specific activity of Ngo-Lig E on different double-stranded breaks. (C) pH dependence of Ngo-Lig E specific activity on singly-nicked DNA. Values represent the percentage of total substrate ligated quantified from band intensities and are the mean of three replicates. Error is the standard error of the mean
Fig. 5 (A) Schematic of FAM-labelled DNA substrates with ligatable breaks, (B) Specific activity of Ngo-Lig E on different double-stranded breaks. (C) pH dependence of Ngo-Lig E specific activity on singly-nicked DNA. Values represent the percentage of total substrate ligated quantified from band intensities and are the mean of three replicates. Error is the standard error of the meanCC BY · PubMed Central
Fig. 6 Structure of Ngo-Lig E. (A) Overall structure of DNA-engaged Ngo-Lig E coloured by domain shown as a cartoon (i) or as a surface (ii). (B) View of the NTase domain active site including the adenylated catalytic lysine residue. (C) Position of the disulphide bond in the OB domain. (D) Inter-domain interactions surrounding the linker. Electrostatic and polar interactions less than 5Å are indicated by dashed yellow lines. (E) Interaction of Ngo-Lig E with DNA duplex (i), specific interactions between conserved residues and the ‘complement’ strand of the duplex (ii), specific interactions between residues of the OB domain and the equivalent of the ‘nicked’ strand where the DNA break is located in other DNA-bound ligase structures (iii)
Fig. 6 Structure of Ngo-Lig E. (A) Overall structure of DNA-engaged Ngo-Lig E coloured by domain shown as a cartoon (i) or as a surface (ii). (B) View of the NTase domain active site including the adenylated catalytic lysine residue. (C) Position of the disulphide bond in the OB domain. (D) Inter-domain interactions surrounding the linker. Electrostatic and polar interactions less than 5Å are indicated by dashed yellow lines. (E) Interaction of Ngo-Lig E with DNA duplex (i), specific interactions between conserved residues and the ‘complement’ strand of the duplex (ii), specific interactions between residues of the OB domain and the equivalent of the ‘nicked’ strand where the DNA break is located in other DNA-bound ligase structures (iii)CC BY · PubMed Central
Fig. 7 (A) Sequence alignment of other Lig E proteins that have been structurally characterised from Alteromonas mediterranea (Ame-Lig E) and Psychromonas sp. SP041 (Psy-Lig E). (B) Ngo-Lig and Ame-Lig structures superimposed by alignment of the OB domains. (C) Ngo-Lig and Psy-Lig structures superimposed by alignment of the OB domains. DNA shown is from the Ngo-Lig E structure
Fig. 7 (A) Sequence alignment of other Lig E proteins that have been structurally characterised from Alteromonas mediterranea (Ame-Lig E) and Psychromonas sp. SP041 (Psy-Lig E). (B) Ngo-Lig and Ame-Lig structures superimposed by alignment of the OB domains. (C) Ngo-Lig and Psy-Lig structures superimposed by alignment of the OB domains. DNA shown is from the Ngo-Lig E structureCC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Pan, J., Hicks, J., & Williamson, A.
Affiliations
University of Waikato (NZ)
Corresponding author
Williamson, Adele
ORCID
Venue
BMC Microbiology
Publisher
Research Square Platform LLC
Published
2023-10-06
Volume
24
Issue
1
Pages
29-29
ISSN
1471-2180 (ISSN-L)
DOI
10.21203/rs.3.rs-3396984/v1
PMC
PMC10799422
OpenAlex
W4391052468
Cited by
9
References
1
Citations by year
2026: 4 · 2025: 3 · 2024: 2
FWCI
2.39
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Microbiology (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Australian Nuclear Science and Technology Organisation
  • University of Waikato
  • Australian Cancer Research Foundation
  • Australian Synchrotron
  • Health Research Council of New Zealand (21/754, 23/534)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
Keywords
ATP-dependent DNA ligase, Neisseria gonorrhoeae, biofilm formation, periplasmic enzymes, crystal structure
MeSH terms
DNA Ligase ATP, DNA, Humans, Neisseria gonorrhoeae, DNA Ligases, Biofilms

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