Article Figures & Data
Figures
- FIG 1
Identification of hypermotile variants from A. baumannii ATCC 17978 WT, ΔqseBC, and ΔygiW strains after desiccation stress. (A) Desiccation survival was determined by enumeration of viable cells (CFU/ml) over a 30-day period. Markers represent mean values of viable cells and error bars the standard errors of the means calculated on days 0, 1, 3, 5, 7, 9, 15, 21, and 30. Four biological replicates were undertaken over two independent experiments. The pink arrow indicates the day that hypermotile variants were identified. (B) Images of hypermotile variants (blue arrows) obtained from rehydrated desiccated cells after ON incubation at 37°C on 1% LB agar.
- FIG 2
Insertions in the hns locus from hypermotile variants and relationship between ISAba12 and MITEAba12. (A) Examples of amplicons generated from PCR across the hns locus from hypermotile isolates compared to the wild type and the previously identified Δhns mutant (27). The amplicon from the ΔygiW Δhns::MITEAba12 strain (663 bp) was 122 bp larger than that from the wild-type control (541 bp), while the ΔygiW Δhns::ISAba12 strain yielded the same size product as the Δhns control (1,590 bp). (B) The open white arrow depicts the hns gene (ACX60_16755) and direction of transcription, and black triangles with green nucleotide sequences represent the TSD for the two integration sites identified previously (29) as well as in this study. The 113-bp MITE is comprised of an 81-bp central region (CR; blue) flanked by 16-bp imperfect inverted repeat sequences (IRL and IRR; purple). //, break in DNA sequence. The novel insertion site/TSD sequences are in pink. The figure is not drawn to scale. (C) Location of MITEAba12 in the A. baumannii ATCC 17978 genome. The 3′ end of ACX60_04650 is fused to MITEAba12, leading to a truncation and the formation of a pseudogene. The deduced amino acid sequence for the modified ACX60_04650 is designated by a single letter code above the underlined nucleotide sequence, and the asterisk indicates the proposed stop codon. Purple and blue nucleotides represent TIR and CR, respectively, of MITEAba12. (D) Nucleotide alignment of 12 bp up- and downstream of the MITEAba12 element in hns of the A. baumannii ATCC 17978 ΔygiW strain, ISAba12 elements present in ATCC 17978, and ISAba12 in hns [ISAba12 (hns)] (27). TIR and TSD are in purple and pink, respectively. Purple underlined nucleotides represent the mismatching base in IRR. The black bracket indicates the size of the region between IRL and IRR, either 81 bp for MITEAba12 or up to 1,008 bp for ISAba12.
- FIG 3
Nucleotide alignment of all MITEAba12 elements identified in this study. The nucleotide sequence above the alignment (black box) denotes the consensus sequence, MITEAba12(c), derived using WebLogo software (35). MITEAba12 sequences with nucleotide variations are displayed. Subgroup representatives are numbered and in boldface type with numbers in parentheses indicating the total number of MITEAba12 copies with that sequence. A, T, G, and C nucleotides are denoted in blue, yellow, purple, and green boxes, respectively. Black lines and asterisks represent the terminal inverted repeats (IRL and IRR) and conserved bases, respectively. See Table S1 for a full list of MITEAba12 elements included in each subgroup and Table 1 for strain accession numbers.
- FIG 4
Characterization of target site duplications flanking MITEAba12. (A) Graphical representation of AT richness (%) identified from all target site duplications flanking MITEAba12 elements. (B) Nucleotide logo generated from all target site duplication events using WebLogo software (35).
- FIG 5
MITEAba12 is located within Tn6645 in Moraexella osloensis CCUG 350. Gray arrows indicate the direction of transcription, and blue arrows represent ISAba3 elements forming the boundaries of Tn6645. Identity between regions is indicated by the color gradient. (A) Alignment of nucleotide sequence from AXE82_04585 to AXE82_06445 in M. osloensis CCUG 350 and the corresponding region in strain KSH (73) (GenBank accession numbers CP014234.1 and CP024180.2, respectively). Gene names and locus tags are derived from M. osloensis CCUG 350 annotation. (B) Alignment of Tn6645 from M. osloensis CCUG 350 and part of the A. guillouiae NBRC 110550 chromosome (GenBank accession number AP014630.1 [43]). Identity between Tn6645 and A. guillouiae NBRC 110550 starts 80 bp downstream from the TIR of ISAba11. The 8-bp TSDs flanking Tn6645 are shown. The location of MITEAba12 is indicated by the orange triangle. Sequences were obtained from the NCBI database and aligned and visualized using the Easyfig 2.2.2 tool (74).
Tables
- TABLE 1
Bacterial strains that harbor full-length MITEAba12 elements
Strain or plasmid No. of MITEAba12
elements per strainIsolation source/origin Accession no. and
reference or sourceStrain A. baumannii DS002 22 Soil, India CP027704.1, unpublished A. indicus SGAir0564 10 Air, Singapore CP024620.1 (75) A. johnsonii XBB1a 7 Hospital sewage, USA CP010350.1 (76) A. junii 65 5 Limnetic water, Russia CP019041 (77) Acinetobacter sp. strain SWBY1a 5 Hospital sewage, China CP026616.1, unpublished A. baumannii B8300 4 Human bloodstream,
southern IndiaLFYY00000000.1 (78) Acinetobacter sp. strain ACNIH1 3 Hospital plumbing, USA CP026420.1 (79) A. baumannii ABNIH28 3 Hospital plumbing, USA CP026125 (79) Acinetobacter sp. strain TGL-Y2 2 Frozen soil, China CP015110.1, unpublished A. baumannii B8342 2 Human bloodstream,
southern IndiaLFYZ00000000.1, (80) M. osloensis CCUG 350 1 Human cerebrospinal fluid, USA CP014234.1, unpublished A. haemolyticus TJS01 1 Human respiratory tract, China CP018871.1, unpublished Acinetobacter sp. strain NCu2D-2 1 Murine trachea, Germany CP015594 (81) Acinetobacter sp. strain ACNIH2a 1 Hospital plumbing, USA CP026412.1 (79) A. baumannii ATCC 17978 1 Human meninges, France CP012004.1 (5) A. junii WCHAJ59 1 Hospital sewage, China CP028800.1, unpublished A. baumannii AR_0083 1 Unknown CP027528.1, unpublished Acinetobacter sp. strain WCHA45a 1 Sewage, China CP028561.1, unpublished A. baumannii MADb 1 Human skin, France AY665723.1 (82) Plasmids A. schindleri SGAir0122, pSGAir0122 2 Air, Singapore CP025619.1 (83) A. baumannii A297 (RUH875), pA297-3 1 Human urinary tract, Netherlands KU744946 (46) A. johnsonnii XBB1, pXBB1-9 1 Hospital sewage, USA CP010351.1 (76) A. lwoffii ED45-23, pALWED2.1 1 Permafrost, Russia KX426229 (53) A. baumannii AbPK1, pAbPK1a 1 Ovine respiratory tract, Pakistan CP024577 (84) Acinetobacter sp. strain DUT-2, unnamed 1 1 Marine sediment, China CP014652, unpublished Acinetobacter sp. strain BW3, pKLH207 1 Stream water, USA AJ486856 (85) A. towneri strain G165, pNDM-GJ01 1 Human stool, China KT965092 (86) A. baumannii D46, pD46-4 1 Human urine, Australia MF399199 (52) Acinetobacter sp. strain ACNIH2, pACl-3569 1 Hospital plumbing, USA CP026416.1 (79) Acinetobacter sp. strain WCHA45, pNDM1_100045 1 Hospital sewage, China CP028560.1, unpublished A. baumannii CHI-32, pNDM-32 1 Human bloodstream, India LN833432.1, unpublished A. defluvii WCHA30, pOXA58_010030 1 Hospital sewage, China CP029396.1, unpublished A. pittii WCHAP005069, pOXA58_005069 1 Clinical isolate, China CP026086.1, unpublished A. pittii WCHAP100004, pOXA58_100004 1 Clinical isolate, China CP027249.1, unpublished A. pittii WCHAP005046, pOXA58_005046 1 Clinical isolate, China CP028573.1, unpublished Acinetobacter sp. strain SWBY1, pSWBY1 1 Hospital sewage, China CP026617.1, unpublished - TABLE 2
IS with TIR closely related to those of ISAba12 and MITEAba12
IS namea IRL sequence IRR sequence Length (bp) TSD (bp) MITEAba12 GGCTTTGTTGCACAAA GGCTTTGTTGCATAAA 113 9 ISAba12 GGCTTTGTTGCACAAA GGCTTTGTTGCACAAA 1,039 9 IS17 GGCTTTGTTGCACAAA GGCTTTGTTGCACAAA 1,040 9 ISAba5b GGCTTTGTTGCACAAA GGCTTTGTTGCATAAA 1,044 ND ISAba7 GGCTTTGTTGCATAAA GGCTTTGTTGCACAAA 1,039 9 ISAba10 GGCTTTGTTGCATAAATA GGCTTTGTTGCACAAATA 1,023 9 ISAba13 GGCTTTGTTGCACAAA GGCTTTGTTGCACAAA 1,039 9 ISAba40 GGCTTTGTTGCACAAA GGCTTTGTTGCACAAA 1,039 9 ISAha1 GGCTTTGTTGCACAAAC GGCTTTGTTGCACAAAC 1,039 4 ISAha2 GGCTTTGTTGCACAAA GGCTTTGTTGCACAAA 1,040 ND ISAha3 GGCTTTGTTGCACAAA GGCTTTGTTGCATAAA 1,039 ND ISAjo1 GGCTTTGTTGCACAAA GGCTTTGTTGCATAAA 1,039 3 ISAlw1 GGCTTTGTTGCACAAAG GGCTTTGTTGCACAAAG 1,038 ND ISEcl7 GGCTTTGTTGCACAAA GGCTTTGTTGCATAAA 1,052 9 ISNov2 GGCTTTGTTGCGCAAAT GGCTTTGTTGCATAAAT 1,048 9 - TABLE 3
Strains and plasmids used in this study
Strain or plasmid Genotype or descriptiona Reference or source Strains A. baumannii ATCC 17978 Noninternational type clone (wild type) ATCC (70) ΔqseBC ATCC 17978 with Eryr insertion disruption in qseBC This study ΔygiW ATCC 17978 with Eryr insertion disruption in ygiW This study Δhns ATCC 17978 with hns disrupted by ISAba12 27 Δhns::ISAba12 ATCC 17978 with hns disrupted by ISAba12 This study ΔqseBC Δhns::ISAba12 ΔqseBC with hns disrupted by ISAba12 This study ΔygiW Δhns::ISAba12 ΔygiW with hns disrupted by ISAba12 This study ΔygiW Δhns::MITEAba12 ΔygiW with hns disrupted by MITEAba12 This study Δhns pWH0268 Δhns with pWH0268 27 Δhns::ISAba12 pWH0268 Δhns::ISAba12 with pWH0268 This study ΔqseBC Δhns::ISAba12 pWH0268 ΔqseBC Δhns::ISAba12 with pWH0268 This study ΔygiW Δhns::ISAba12 pWH0268 ΔygiW Δhns::ISAba12 with pWH0268 This study ΔygiW Δhns::MITEAba12 pWH0268 ΔygiW Δhns::MITEAba12 with pWH0268 This study E. coli DH5α λpir F– Φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK, mK+)
phoA supE44 λ– thi-1 gyrA96 relA1 λpir,
conjugative strain which can host λ-pir-dependent plasmids87 Plasmids pAT04 Tetr; pMMB67EH with RecAb system 71 pGEM-T Easy Ampr; T-overhang cloning vector Promega pVA891 Cmlr Eryr; Source of Eryr cassette 88 pWH0268 Ampr; pWH1266 with hns cloned via BamHI restriction site 27 ↵a Abbreviations: Amp, ampicillin; Cml, chloramphenicol; Ery, erythromycin; Tet, tetracycline.
- TABLE 4
Primers used in this study
Primer function and name Sequencea (5′–3′) Reference or source Cloning and sequencing of hns genes
with integrated mobile genetic elementshns_F GAGACATATGATGCATCATCATCATCATCATATAAATATTAAGAAAATATATTA 27 hns_R TCTCGGATCCTTAGATTAAGAAATCTTCAAG 27 M13 F GTAAAACGACGGCCAG Promega M13 R CAGGAAACAGCTATGAC Promega Identification of presence of IS ACX60_04650_F CGTATTTGGGTCTTGGGGAA This study ACX60_04650_R CCTTTGGTAAGTACTTTAT This study ACX60_18935_F AGCAACTGAAGCTGAAATTCG 27 ACX60_18935_R TTGGTTCCGAATTAGACTTGC 27 ACX60_04795_F CAGTCAGGTTCGCCAT This study ACX60_04795_R GACCAGACAATACAATG This study Construction of ΔqseBC and ΔygiW ΔqseBC ΔqseBC_UFR_F CAATTCCGCGATAAGAGC This study ΔqseBC_UFR_R CTATCAACACACTCTTAAGCCTGTTATATCCTGAT This study ΔqseBC_DFR_F CGGGAGGAAATAATTCTATTTGCAGTCACAACTGG This study ΔqseBC_DFR_R GTAGTAACCAGAACAGCAC This study ΔqseBC_NOL_F GGCAAGGACGTCCTGTTT This study ΔqseBC_NOL_R GGGCTGAAAAACTTCAAC This study ΔqseBC_Ery_F CTTAAGAGTGTGTTGATAG 39 ΔqseBC_Ery_R ATAGAATTATTTCCTCCCG 39 ΔygiW ΔygiW_UFR_F CAGTTGAAATGGCATCCATTAC This study ΔygiW_UFR_R CTCTTAAGGTATAGGAACTTCAAAATTACCCTCTGTTA This study ΔygiW_DFR_F GAGGAAATAAGAAGTTCCTATACTAAATTAATTTCTACATTTATTCC This study ΔygiW_DFR_R GAGAGCGGCCGCCTCATTTTAAGTCTCCCATAC This study ΔygiW_NOL_F CGGCATTTATGAGTTTATGCCAG This study ΔygiW_NOL_R GGCTTGCCCCAACTGA This study ΔygiW_Ery F GAAGTTCCTATACCTTAAGAGTGTGTTGATAG This study ΔygiW_Ery R GTATAGGAACTTCTTATTTCCTCCCGTTAAATAATAGATAAC This study ↵a Nucleotides in boldface represent incorporated restriction sites: NdeI, CATATG; BamHI, GGATCC; NotI, GCGGCCGC.
FIG S1
Motility of A. baumannii ATCC 17978 variants and complemented derivatives. Cells grown overnight were used as the inoculum for motility assays on LB medium containing 0.25% agar. WT ATCC 17978, ΔqseBC, and ΔygiW cells displayed a nonmotile phenotype. Derivatives of these strains with an hns gene interrupted by ISAba12 or MITEAba12 and Δhns (27) displayed a hypermotile phenotype, dispersing from the original inoculum site to cover the entire plate surface. Reintroduction of a WT copy of hns on the shuttle vector pWH1266 (pWH0268) returned strains to the parental nonmotile phenotype. Images are a representative example of results obtained. Download FIG S1, TIF file, 1.9 MB.
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TABLE S1
List of MITEAba12 elements and their corresponding strain names that formulate MITEAba12 subgroups 1 to 10. Download Table S1, DOCX file, 0.01 MB.
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FIG S2
Putative σ70 promoter sequences identified in MITEAba12(c). The σ70 promoter sequences were predicted using the Softberry BPROM tool. (A) Pink and blue nucleotide sequences represent outward-facing promoters, reading out through the IRL and IRR sequences (Pout IRL and Pout IRR, respectively), and the orientation of transcription is shown by arrows. Nucleotides underlined and double underlined denote −10 box and −35 box sequences, respectively. Purple nucleotides denote IRL and IRR, with putative translational start codons in boldface with their corresponding putative ribosome binding sites (RBS) shaded in pink and blue, respectively. (B) Alignment of the putative Pout IRL and Pout IRR and RBSs in MITEAba12(c) against the E. coli σ70 promoter and RBS consensus and Pout of ISAba1 coupled with the adjacent region and RBS in the ISAba1-activated blaampC gene of A. baumannii CLA-1 (38). The nucleotide length between the −10 and −35 boxes [Sep. (bp)] is indicated. Download FIG S2, TIF file, 1.5 MB.
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This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
