1. Dr. Lori Snyder, Reader in Biotechnology, Kingston University, London, UK. L.Snyder@kingston.ac.uk
Firat Elbeyioglu, First Class MSc in Biochemistry with Honours, Kingston University 2012. f_e@hotmail.co.uk
Sabrina Roberts, 1st year PhD student, Kingston University. Completing in 2014. k0610485@kingston.ac.uk
Marta Zelewska, First Class MSc in Biochemistry with Honours, Kingston University 2012. marta.a.zelewska@gmail.com
Madhuri Pulijala, MSc in Biotechnology with Distinction, Kingston University 2011. pretty.madhu@gmail.com
Russell Spencer-Smith, First Class MSc in Biochemistry with Honours, Kingston University 2010, MSc by Research, Kingston
University 2011, first year PhD student at the University of Illinois at Chicago with Vadim Gaponenko. russell_spencer@ymail.com
Movement of Correia Repeat Enclosed Elements
during laboratory culture
Firat Elbeyioglu, Sabrina Roberts, Marta Zelewska, Madhuri Pulijala,
Russell Spencer-Smith & Lori Snyder*
School of Life Sciences, Kingston University, UK
Abstract
The Correia Repeat Enclosed Element, an
IS-like element, has been predicted to be
mobile within the gonococcal genome.
Although there is evidence of ancestral
movement of these elements, no previous
study has provided evidence for current
mobilisation. Previous studies have
compared the genomic locations of Correia
Repeat Enclosed Elements in the Neisseria
spp., demonstrating that otherwise identical
regions in the same species have either a
Correia Repeat Enclosed Element or the
target TA insertion site. In this study we
report for the first time movement of Correia
Repeat Enclosed Elements, through
inversion of the element at its chromosomal
location. Analysis of Ion Torrent generated
genome sequence data from Neisseria
gonorrhoeae strain NCCP11945 passaged
for 8 weeks in the laboratory under standard
conditions and stress conditions revealed a
total of 38 inversions: 25 were exclusively
seen in the stressed sample; 8 in the control
sample; and the remaining 5 were seen in
both samples. The sequence read data
provided evidence for their mobility during
laboratory culture, through inversion rather
than translocation. These inversions have
the capability to alter gene expression in N.
gonorrhoeae through the previously
determined activities of the sequence
features of these elements.
Materials and Methods
N. gonorrhoeae strain NCCP11945 was
grown in normal culture on GC agar (Oxoid)
with Kellogg glucose and iron supplements
at 37°C in a candle tin for 8 weeks, with
passage to fresh media every two days. A
parallel stress culture contained a sublethal
concentration of nalidixic acid (128 mg/ ml).
DNA was extracted from the normal and
stress culture samples using the Gentra
Puregene Yeast/Bact. Kit (Qiagen) according
to manufacturer’s instructions.
Next-generation genome sequencing of 1 μg
of the extracted DNA used the Ion Personal
Genome Machine, Ion Express Fragment
Library kit, Ion Express Template kit, and Ion
Sequencing kit (Life Technologies).
The sequence read data were aligned
against the N. gonorrhoeae NCCP11945
genome sequence (CP001050)12 using CLC
Genomics Workbench version 5 software
and manually analysed for CREE
movements. CREE locations in this genome
were reported previously4. Identification of
potential promoter regions used data
reported previously4,9.
Exclusive to the Neisseria spp., Correia Repeat Enclosed Elements (CREE)
resemble IS elements1-3, are in different locations in different strains3-5, and
contain IHF binding sites5-6, promoters7-9, and RNase III processing sites10-11.
But, can they move?
References1Correia et al., J Bacteriol. 167(3), 1009-1015, 1986.
2Correia et al., J Biol Chem. 263(25), 12194-12198, 1988.
3Liu et al., J Bacteriol.184(22), 6163-6173, 2002.
4Snyder et al., BMC Genomics 10, 70, 2009.
5Buisine et al., FEBS Lett. 522(1–3), 52-58, 2002.
6Rouquette-Loughlin et al., Mol. Microbiol. 54:731–741, 2004.
7Black et al., J Bacteriol. 177(8), 1952-1958, 1995.
8Snyder et al., Mol Microbiol. 47(2), 431-442, 2003.
9Siddique et al., PLoS Genetics. 7, 1, 2011.
10De Gregorio et al., Biochim Biophys Acta. 1576(1–2), 39-44, 2002.
11De Gregorio et al., Biochem J. 374(Pt 3), 799-805, 2003.
12Chung et al., J Bacteriol. 190(17), 6035-6036, 2008.
Figure 2: Evidence of inversion of a 71 bp CREE.
As in Figure 1, the read data is aligned against the
reference genome sequence data. For this 71 bp CREE,
from position 378,769 to 378,839, there are inverted
repeats, indicated by the green arrows, and a core region
that has directionality, indicated by the blue arrow. Some of
the reads align with the genome sequence (small red
arrows), but most of the reads show an inverted orientation
to the CREE. Mismatches in the read data are shown in
lighter green and lighter red. Note the mismatches in the
black boxes match the reverse complement of the genome
sequence. The whole of the CREE has inverted.
Figure 3: 38 out of 131 CREE have inverted.
Graphical representation of the sequence data observed:
CREE orientation for the reference genome (green);
sequencing reads with the CREE in the genome sequence
orientation (blue); and sequencing reads with the CREE in
an inverted orientation (red). Of the 131 CREE in N.
gonorrhoeae strain NCCP11945, 93 are in the genome
sequence orientation and 38 show evidence of inversion.
Figure 4: Inversion of CREE promoters.
Graphical representation of the CREE in position 2,186,864
to 2,186,967, with promoter orientation before and after an
inversion. The reference genome sequence orientation
(green), aligned reads (blue), and inverted reads (red),
show that the associated Black promoter7 either will not
(green & blue) or will (red) drive transcription of NGK_2601,
annotated as encoding cadmium resistance protein CadD.
Twelve of the 38 inverted CREE appear to have promoters.
Figure 1: Each CREE location was manually analysed.
Shown here is the reference genome sequence15, labelled
CP001050, and each individual sequencing read from the
Ion Torrent Personal Genome Machine in green (forward
reads) and red (reverse reads). The CREE is represented by
the red arrow and delineated with the vertical red lines. For
the 106 bp CREE shown here, from position 1,115,108 to
1,115,213, there are no signs of movement. The read data
before and after the CREE aligns with the chromosomal
location, reads that go through the CREE match this location
at both ends, and the CREE itself aligns well.
Table. All CREE with
evidence of inversion.
Conclusions
• CREE of all sizes are capable of movement via
inversion during normal culturing in the laboratory.
• CREE appear to invert more frequently under stress
conditions (Control = 13; Test =30).
• Cultures contain mixed populations in which some
CREE in some cells have inverted.
• Inversion of CREE promoters may be a switchable
gene expression mechanism.
• Movement from one location in the chromosome to
another, like an IS element, was not observed.
start stop
13475 13580 106 Control
137920 138026 107 Control
210653 210757 105 Test
315775 315881 107 Both
332258 332361 104 Test
378769 378839 71 Test
409189 409295 107 Both
497451 497605 155 Test
518804 518908 105 Test
625567 625673 107 Both
636542 636648 107 Test
665821 665926 106 Control
684759 684863 105 Test
748921 749074 154 Test
773275 773380 106 Both
782431 782536 106 Test
933079 933183 105 Control
949754 949858 105 Test
958334 958439 106 Control
1033492 1033597 106 Test
1073168 1073274 107 Control
1321069 1321174 106 Test
1324830 1324935 106 Test
1465780 1465934 155 Test
1519344 1519497 154 Test
1577266 1577372 107 Test
1753230 1753334 105 Control
1882841 1882913 73 Test
1974277 1974383 107 Both
1977219 1977328 110 Test
2059092 2059198 107 Test
2106628 2106696 69 Test
2123050 2123202 153 Test
2151965 2152118 154 Test
2161831 2161937 107 Test
2172837 2172941 105 Test
2186864 2186967 104 Control
2189554 2189660 107 Test
size
(bp)
Inverted
sample
CREE location