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<title>Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus</title>
<meta name="Subject" content="BMC Evolutionary Biology 2010 10:230. doi:10.1186/1471-2148-10-230"/>
<meta name="Author" content="Muktak Aklujkar"/>
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Aklujkar and Lovley BMC Evolutionary Biology 2010, 10:230
http://www.biomedcentral.com/1471-2148/10/230
RESEARCH ARTICLE
Open Access
Interference with histidyl-tRNA synthetase by
a CRISPR spacer sequence as a factor in the
evolution of Pelobacter carbinolicus
Muktak Aklujkar*, Derek R Lovley
Abstract
Background: Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or
produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P.
carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer
sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing
identical sequences.
Results: CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P.
carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous
to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited
growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P.
carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient
for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination
during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral
genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an
explanation for its physiological differences from other Geobacteraceae.
Conclusions: The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring
ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition
that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR
spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer
from one species in the context of repeats of another species, and the first report of a potential impact of CRISPR
on genome-scale evolution by interference with an essential gene.
Background
Clustered regularly interspaced short palindromic
repeats (CRISPR), which consist of direct repeats of a
short sequence (21-47 bp) separated by nonrepetitive
sequences of similar size, have been identified in the
genome sequences of almost all archaea and numerous
bacteria, with a variable complement of adjacent
CRISPR-associated (cas) genes [1-9]. A fraction of the
spacer sequences between repeats have been found to
match sequences termed “proto-spacers” within genes,
from which they may be derived [8,10,11], and the fact
* Correspondence: [email protected]
University of Massachusetts Amherst, Amherst, MA, 01003, USA
that many of these genes belong to phage or plasmid
entities led to the hypothesis that CRISPR and the Cas
proteins may function as an RNA interference-based
immune system [6]. The link between specific CRISPR
spacers and proto-spacers and phage resistance has been
established by mutational analysis in Streptococcus thermophilus [12,13], and by testing synthetic CRISPR constructs in Escherichia coli [14]. Similarly, resistance of
Staphylococcus epidermidis to a conjugative plasmid has
been shown to depend on a CRISPR spacer and the corresponding proto-spacer [15]. Expression of CRISPR loci
as long transcripts processed into smaller RNA molecules has been observed in several archaea [5,16-19] and
bacteria [14,20]. A complex of Cas proteins has been
© 2010 Aklujkar and Lovley; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Aklujkar and Lovley BMC Evolutionary Biology 2010, 10:230
http://www.biomedcentral.com/1471-2148/10/230
shown to carry out this processing in E. coli and to be
required for resistance to infection [14]; a different protein (Cas6) processes CRISPR transcripts in Pyrococcus
furiosus [21]. CRISPR-derived RNAs have been shown
to form RNA-protein complexes in P. furiosus [19],
which leads to degradation of RNAs containing matching proto-spacers [22], whereas DNA was shown to be
the target of interference by spacer-containing RNAs in
S. epidermidis [15]. Although CRISPR are widely
regarded as an immunological phenomenon, CRISPR and
cas genes have also been implicated in spore development of Myxococcus xanthus [3,23] and in inhibition of
biofilm formation and swarming of Pseudomonas aeruginosa by a lysogenic phage [24], and there has been speculation that spacers with matches to housekeeping genes
represent a novel mechanism of gene regulation [25].
The Geobacteraceae, a Fe(III)-respiring family of Deltaproteobacteria, are of interest for their role in bioremediation of U(VI)-contaminated environments and
their ability to donate electrons directly to graphite electrodes, producing an electrical current [26,27]. Pelobacter carbinolicus is a member of the Geobacteraceae that
grows by fermentation of acetoin and 2,3-butanediol, as
well as by indirect Fe(III) respiration with ethanol as the
electron donor and acetate as the end product [28,29].
Unlike its relatives in the genus Geobacter, P. carbinolicus cannot reduce Fe(III) directly in the absence of sulfur or sulfide [30], or produce electricity [31]. The
genome of P. carbinolicus was sequenced for the purpose of comparison to those of Geobacter species, three
of which have been extensively curated: Geobacter sulfurreducens [32], Geobacter metallireducens [33] and
Geobacter bemidjiensis (Aklujkar et al., submitted). This
report explores how evolution of the P. carbinolicus
genome may have been influenced by a spacer within
the CRISPR locus that matches a proto-spacer within
histidyl-tRNA synthetase (hisS), resulting in the elimination of ancestral genes containing multiple closely
spaced histidines. The interfering nature of the spacer
was confirmed by introducing it to a transgenic G. sulfurreducens strain containing the target gene.
Methods
Analysis of CRISPR spacers
The CRISPR locus was identified when manual curation
of the P. carbinolicus genome revealed a series of suspiciously repetitive predicted genes. The nonredundant
nucleotide sequence database was queried with each of
the 111 CRISPR spacers of P. carbinolicus using the
BLAST algorithm [34], with the minimum possible
word size of 7 bp and without filtering out low-complexity regions of the queries. Alignments with five or
fewer mismatches out of 32 bases were considered
significant.
Page 2 of 15
Phylogenetic analysis of HisS and HisZ proteins
The sequences of all predicted hisS gene products of the
Geobacteraceae, together with HisS and HisZ protein
sequences representative of various families of Bacteria
and Archaea, were aligned by TCoffee [35] and trimmed
using Mesquite (Maddison, W. P., and Maddison, D. R.
2006. Mesquite: a modular system for evolutionary analysis. Version 1.12). Phylogenetic trees were constructed
using Phylip (Felsenstein, J. 2005. PHYLIP (Phylogeny
Inference Package) version 3.6) with 500 bootstrap runs.
Quantitative real-time PCR of reverse-transcribed RNA
P. carbinolicus strain DSM2380 was grown as previously
described [36] with ethanol as the electron donor and
Fe(III) as the electron acceptor. RNA was isolated from
triplicate chemostat cultures as previously described
[37,38]. Transgenic G. sulfurreducens strains were grown
in NBAF medium [39] and RNA was isolated from
actively growing triplicate batch cultures at an OD600 of
0.20 to 0.31. The absence of DNA contamination was
confirmed by PCR as previously described [36] with primer pairs specific for CRISPR spacer #1, for hisS and
for hisZ (Table 1), using P. carbinolicus or G. sulfurreducens genomic DNA (isolated with the MasterPure
DNA Purification Kit from EPICENTRE Biotechnologies, Madison, WI) as a control. Six to twelve clones of
each genomic DNA PCR product were sequenced to
verify the specificity of the primers. Reverse transcription was performed with the Enhanced Avian First
Strand Synthesis Kit (Sigma-Aldrich, St. Louis, MO) as
described previously [37], using each primer individually
at 2 μM concentration with 400 ng of RNA in 20 μl
total volume. Successful reverse transcription and the
feasibility of DNA amplification in the presence of RNA
were verified by PCR using 5 μl of this reaction. Quantitative real-time PCR (QRT-PCR) was performed with
two to four technical replicates (9.5 μl of a tenfold dilution of cDNA, corresponding to 19 ng of RNA) for each
of three biological replicates in a Taqman 7500 instrument using 2 × Power SYBR Green PCR master mix
(Applied Biosystems, Foster City, CA) and primer pairs
at 9 nM concentration in 25 μl total volume, for 50
cycles with an annealing temperature of 60°C and triplicate standards of spacer #1 and hisS PCR products from
P. carbinolicus genomic DNA and a hisZ PCR product
from G. sulfurreducens genomic DNA, encompassing
four orders of magnitude.
Recombinant DNA techniques
All restriction enzymes were purchased from New England Biolabs; LA Taq polymerase was from Takara
Mirus Bio; plasmids were propagated in E. coli TOP10
cells from Invitrogen; DNA purification kits for plasmids
and agarose gel slices were from QIAGEN, and the
Aklujkar and Lovley BMC Evolutionary Biology 2010, 10:230
http://www.biomedcentral.com/1471-2148/10/230
Page 3 of 15
Table 1 Oligonucleotides for QRT-PCR and genetic manipulations.
Primers for QRT-PCR
Name
MA0326
Purpose
Location
Sequence (5’ to 3’)
PCR of spacer #1
spacer #2
CCTGGTTGAGGTTAGCGTTGA
outside CRISPR
AATTCGGTGGCCAGTTGTTC
MA0327
MA0328
PCR of hisS Pcar_1041
MA0441
PCR of hisZ GSU3307
CAGGAAGCCACCAAGGAT
antisense strand
MA0329
sense strand
TGGGAGCCGAGTTGATTG
sense strand
CAAACTGATTGCCGTTCCTT
antisense strand
MA0442
AGGCCGATGAGTTCTACGC
Primers for construction of hisS transgenic strain MA159
Name
MA0330
Purpose
PCR on 5’ side of hisS GSU1659
MA0331
MA0332
PCR on 3’ side of hisS GSU1659
GTACGTTCATGATTACAAACTAGTGCTAGCATAGCAATAC
CTGCATTG
AGTCCATTCCTCCTGTGG
PCR of hisS Pcar_1041
MA0335
MA0052
TGACATCTCGCTGGACCGGG
CTATGCTAGCACTAGTTTGTAATCATGAACGTACCTACTC
CTTTAATTG
MA0333
MA0334
Sequence (5’ to 3’)
AAGGGATCTATCATGAGCATATCAGGCATTAAGGG
GCGCGGCGCGACTAGTTTCCTCGTGTCTTTTCC
gentamicin marker
MA0053
TGCATATGGCTCTAGAATAACTTCGTATAGC
TCGATAAGCTTCTAGAATAACTTCGTATAATG
Oligonucleotides for construction of chimeric CRISPR expression plasmid pMA35
Name
MA0269
Purpose
PCR of lacI-taclacUV5 promoter
MA0270
MA0429
GCATGCGTGTGAAATTGTTATCCGC
syntheticCRISPR of spacer #1
MA0430
MA36R
Sequence (5’ to 3’)
ACATGTCACTGCCCGCTTTCCAGTC
AATTCGGTTCATCCCCGCGCATGCGGGGAACACATACAT
GAGGGCAAACGCCTTTTGGCCGGCGGCGGTTCATCCCCG
CGCATGCGGGGAACACG
GATCCGTGTTCCCCGCATGCGCGGGGATGAACCGCCGCC
GGCCAAAAGGCGTTTGCCCTCATGTATGTGTTCCCCGCAT
GCGCGGGGATGAACCG
sequencing
CGACATCATAACGGTTC
Note: Within the sequence of the chimeric CRISPR, a single base pair (underlined) has been duplicated in plasmid pMA35-!, at the exact centre of spacer #1.
MasterPure kit for genomic DNA extraction was from
EPICENTRE. To construct a transgenic strain of G. sulfurreducens in which the native hisS gene was replaced
with hisS from P. carbinolicus, three primer pairs (Table
1) were used to amplify the 5′-side and 3′-side flanking
regions of hisS GSU1659 and the coding sequence of
hisS Pcar_1041. The two flanking regions were digested
with Spe I and ligated; this product and the Pcar_1041
amplicon were separately TOPO-cloned (Invitrogen)
and sequenced. Digestion with BspH I (overlapping the
start codon) and Spe I (overlapping the stop codon) was
used to insert the Pcar_1041 gene between the flanking
regions of GSU1659. As a selectable marker, a gentamicin resistance cartridge was amplified from plasmid
pCM351 [40] with Xba I site-containing primers (Table
1), maintained as a TOPO clone, and ligated into the
Nhe I site between the Spe I site and the 3′ flanking
region of GSU1659. (A similar construct, in which only
the marker was inserted without Pcar_1041, was used in
unsuccessful attempts to delete GSU1659.) The entire
hisS replacement construct was excised using EcoR I,
purified from an agarose gel, and electroporated into the
wild type G. sulfurreducens strain DL1 as previously
described [39]. An isolated gentamicin-resistant colony
was streaked for purity before transfer to liquid. The
genotype of this strain, called MA159G, was confirmed
by PCR of genomic DNA, with primers MA0334 and
MA0335 (which amplify hisS Pcar_1041 but not hisS
GSU1659) as well as MA0330 and MA0333 (which give
a larger product for MA159G than for DL1, due to the
inserted marker). The marker, which had loxP sites on
either side, was removed from the chromosome of strain
MA159G by introducing the Cre recombinase expression plasmid pCM158 [40] by electroporation and
selecting for resistance to kanamycin (Sigma). Four colonies of the resultant strain called MA159 were streaked
Aklujkar and Lovley BMC Evolutionary Biology 2010, 10:230
http://www.biomedcentral.com/1471-2148/10/230
Page 4 of 15
for purity and their genotypes were confirmed by PCR;
amplicons were digested with Pst I to distinguish
Pcar_1041 from GSU1659. This strain was electroporated with a plasmid called pRG6 (R. Glaven, personal
communication), which is incompatible with pCM158
and selectable with spectinomycin (Sigma); it differs
from pRG5 [41] only in that it carries the lacI repressor
gene. The chromosomal genotype of this strain was confirmed by PCR and Pst I digestion.
A plasmid vector called pMA36, incompatible with
pRG6, was constructed for isopropylthio-b-D-galactopyranoside (IPTG)-inducible expression of a chimeric
CRISPR containing spacer #1 from P. carbinolicus
between two repeats typical of the CRISPR2 locus of
G. sulfurreducens. The lacI repressor gene and taclacUV5 promoter of plasmid pCD341 [42] were amplified
by PCR with Pci I and Sph I site-containing primers
(Table 1), TOPO-cloned and sequenced, and excised for
ligation into plasmid pCM66 [43], resulting in plasmid
pMA36. The chimeric CRISPR, consisting of annealed
oligonucleotides MA0429 and MA0430 (Table 1), was
ligated between the BamH I and EcoR I sites of pMA36.
The sequence of this plasmid, called pMA35-1, was confirmed using the sequencing primer MA36R (Table 1).
Serendipitously, two variants were discovered: pMA35-2
in which the chimeric CRISPR had expanded to two
copies of spacer #1 with a third copy of the repeat
between them, and pMA35-! in which spacer #1 was
disrupted by duplication of a single G:C base pair at its
exact centre (underlined in Table 1). All three chimeric
CRISPR expression plasmids were electroporated into
DL1 and MA159(pRG6), in parallel with pMA36 as a
control. The genotypes of multiple kanamycin-resistant
colonies of each transformation were confirmed by PCR
of Pcar_1041 followed by Pst I digestion as well as cloning and sequencing, and by sequencing of plasmids present in the genomic DNA extracts (after transformation
into E. coli to improve DNA quality). Another variant of
pMA35-1 called pMA35-0 was serendipitously discovered in which spacer #1 had been deleted by recombination of the repeats on either side.
Bioinformatics
Growth conditions
Quantitative detection of CRISPR spacer #1 transcripts
Transformants of G. sulfurreducens were selected on
NBAF medium [39] containing 1.5% Agar Noble
(Difco), supplemented with 5 mM cysteine hydrochloride and 0.1% yeast extract in an anaerobic chamber.
Growth experiments were carried out with liquid cultures in either NBAF medium supplemented with 1 mM
cysteine hydrochloride or FWAFC medium [39] modified to contain 10 mM acetate and supplemented with
1 mM ferrous ammonium sulfate, in an atmosphere of
N2 and CO2 (80%:20%) in rubber-stoppered 26 ml glass
tubes at 30°C.
In an attempt to determine whether spacer #1 is transcribed into RNA that could have interfered with the
hisS gene at one time, and which strand of trailer end
RNA is predominant in P. carbinolicus, two oligonucleotide primers were designed flanking spacer #1 (Figure 1,
Table 1): MA0326 within spacer #2 and MA0327 just
outside the outermost repeat of the CRISPR. Reverse
transcription of P. carbinolicus RNA into cDNA was
attempted with each single primer, followed by quantitative real-time PCR amplification with both primers. The
amount of spacer #1-containing RNA including the
Codon usage was determined using the CodonFrequency algorithm of the Genetics Computer Group
Wisconsin Package version 10.3 (Accelrys Inc., San
Diego, CA). A script to compute the number of histidines and the distances between them for every protein
sequence in a list was written in Perl.
Results
The CRISPR locus of P. carbinolicus includes a spacer
matching its own histidyl-tRNA synthetase
During manual curation of the P. carbinolicus genome
annotation, the CRISPR locus was identified as 112
repeats of the sequence 5′-GAGTTCCCCGCAGATGCGGGGATGAACCG-3′ (bases in bold predicted
to form a hairpin), separated by spacer sequences of 32
bp (Figure 1). This repeat sequence belongs to phylogenetic cluster 2 of the CRISPR classification system [44]
and the adjacent cas genes (Figure 1) are of the subtype
“Ecoli” [3]. The nonredundant nucleotide sequence database was queried in an attempt to identify genes from
which the 111 CRISPR spacers of P. carbinolicus might
be derived. The only hits with five or fewer mismatched
bases were hits with zero mismatches within the P. carbinolicus genome itself: spacer #1, located at the “trailer”
end of the locus, farthest from the AT-rich “leader
sequence” and cas genes encoding CRISPR-associated
proteins (Figure 1), matched a sequence within the histidyl-tRNA synthetase (hisS) gene Pcar_1041 (Figure 2);
spacer #43 matched the adjacent spacer #44; and spacer
#28 matched the nonadjacent spacer #50. Spacer #1 is
likely to be the oldest spacer because new spacers are
added next to the leader sequence upon exposure of
streptococci to bacteriophage [12,13,45-47], and closely
related strains of bacteria and archaea contain identical
spacers only near the trailer ends of their CRISPR
[2,5,11,18,45,48-51]. This observation led to the hypothesis that P. carbinolicus has experienced interference
with the hisS gene, encoding an essential housekeeping
enzyme, over a significant period of its evolutionary
history.
Aklujkar and Lovley BMC Evolutionary Biology 2010, 10:230
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Page 5 of 15
QRT-PCR amplicon
of spacer #1
cas genes
primer MA0326
primer MA0327
AT-rich “leader”
sequence of CRISPR
noncoding “trailer”
sequence
Pcar_0956
Pcar_0957
cas3
cse1
Pcar_0958 Pcar_0959
cse2
cse4
Pcar_0960 Pcar_0961
cas5e
cse3
Pcar_0964 Pcar_0965
cas1
cas2
Figure 1 The CRISPR locus of P. carbinolicus. This locus consists of 112 repeats (black diamonds) separated by 111 nonrepetitive spacers
(white rectangles). Spacer #1 is at the trailer end, farthest from the cas genes and the AT-rich leader sequence near which new spacers are
typically inserted. Primers MA0326 and MA0327 are based on sequences surrounding spacer #1, and were used to detect its RNA transcript. The
arrangement of the cas genes (located immediately to the left of the leader sequence) is illustrated in the lower half of the figure. The two
intervening genes encode a putative toxin (Pcar_0962) and transcriptional regulator or antitoxin (Pcar_0963).
(a)
hisS gene
sense primer MA0328
(b)
proto-spacer
antisense primer MA0329
QRT-PCR
amplicon
(c)
predicted structure of processed spacer #1 RNA
5’-AUGAACGGCCGCCG-CUCAUGUAUGUGGUCCCCG-3’
G-C
C C
C-G
A-U
A-U
A-U
A
G
G C
G
processed spacer #1 RNA
5’-AUGAACGGCCGCCGGCCAAAAGGCGUUUGCCCUCAUGUAUGUGGUCCCCG-3’
||||||||||||||||||||||||||||||||
3’-GACCGGCGGGACCCGCTTCGGCGGCCGGTTTTCCGCAAACGGGAGTACATAGCGGACGTCGCTGAGCTG-5’
5’-CTGGCCGCCCTGGGCGAAGCCGCCGGCCAAAAGGCGTTTGCCCTCATGTATCGCCTGCAGCGACTCGAC-3’
LeuAlaAlaLeuGlyGluAlaAlaGlyGlnLysAlaPheAlaLeuMetTyrArgLeuGlnArgLeuAsp
proto-spacer within hisS DNA sequence and corresponding HisS amino acid sequence
Figure 2 CRISPR spacer #1 matches a nucleotide sequence within the hisS gene. (a) hisS consists of a catalytic domain (dark grey) and an
anticodon loop recognition domain (light grey) connected by a linker (white stripe). The proto-spacer sequence matching spacer #1 (black
stripe) is within the anticodon loop recognition domain. Primers MA0328 and MA0329 were designed to amplify a cDNA segment from the
catalytic domain. (b) Predicted secondary structure of a processed CRISPR transcript (initiated at the leader sequence) that contains spacer #1,
before hybridization to hisS DNA. Sequences from the repeats flanking spacer #1 are underlined. (c) Predicted hybridization of a proto-spacer
segment within the anticodon loop recognition domain of hisS DNA (template strand) with a processed spacer #1 RNA. The proto-spaceradjacent motif CTT is shown in bold.
Aklujkar and Lovley BMC Evolutionary Biology 2010, 10:230
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sequence found on the sense strand of hisS, detected by
primer MA0327, was not significantly different from the
amount of spacer #1-containing RNA corresponding to
the antisense of hisS, detected by primer MA0326
(Figure 3). Control PCR reactions without reverse transcription yielded no product, indicating that DNA contamination was negligible and only RNA of both strands
was detected.
The sense strand spacer #1-containing RNA detected in
this experiment may represent the 3′ end of a long transcript initiated near the leader sequence, whereas the
antisense strand spacer #1-containing RNA may be produced independently from a promoter at the opposite
end of the CRISPR. It is also possible that one strand is
produced from the other by an unidentified RNA-directed RNA polymerase. If the sense strand spacer #1-containing RNA undergoes processing similarly to the
E. coli CRISPR transcript [14], which belongs to the
same phylogenetic cluster of repeat sequences as the
P. carbinolicus CRISPR [44], cleavage within the stemloops of the repeats flanking spacer #1, followed by 3′
end trimming, would release a short RNA with predicted secondary structure (Figure 2b). This or the corresponding antisense strand spacer #1-containing RNA
molecules per ng
1.2 x 107
0.8 x 107
0.4 x 107
0
MA0327 MA0326
Figure 3 Spacer #1 is transcribed into RNA in P. carbinolicus,
with both strands similarly abundant. Reverse transcription was
performed with either primer MA0327 (grey bar) or primer MA0326
(white bar), and the amount of cDNA was quantified by QRT-PCR.
The mean of three biological replicates is shown; error bars
represent the minimum and maximum.
Page 6 of 15
may hybridize to the proto-spacer DNA sequence within
the hisS gene (Figure 2c).
Phylogenetic and experimental evidence that
interference with hisS cannot be compensated
It is surprising that spacer #1 is retained by P. carbinolicus if it interferes with the essential function of histidine
activation for protein synthesis. Comparative genome
analysis revealed that P. carbinolicus and its close
relatives, the Geobacteraceae, possess two full-length
hisS-like genes, whereas other bacteria have only one.
Interference with Pcar_1041 by spacer #1 might have
had negligible effect if Pcar_0202 also produced histidyltRNA synthetase activity. However, both phylogenetic
and mutational studies suggest that Pcar_1041 is essential, being the only real hisS, as detailed below.
In some bacteria there is a hisS-related gene called
hisZ, which produces a protein that lacks the C-terminal
anticodon loop recognition domain of a true histidyltRNA synthetase, functioning instead as a regulatory
subunit of ATP phosphoribosyltransferase, the first
enzyme of histidine biosynthesis [52]. In bacteria that
possess hisZ, the hisG gene encoding the catalytic
domain of ATP phosphoribosyltransferase is shorter
than in bacteria that do not possess hisZ [53]. A short
hisG gene is present in P. carbinolicus and all other
Geobacteraceae, but unlike previously described hisZ
genes, both hisS-like genes contain obvious anticodon
loop recognition domains. Phylogenetic analysis showed
that the Pcar_1041 gene product and orthologous protein sequences of Geobacteraceae cluster among the
HisS proteins of other bacteria, whereas the Pcar_0202
gene product and its orthologs belong among the HisZ
proteins (Figure 4). Furthermore, the ortholog of
Pcar_0202 in G. sulfurreducens (GSU3307) could be
deleted (Aklujkar and Lovley, manuscript in preparation), whereas the ortholog of Pcar_1041 (GSU1659)
could only be replaced with Pcar_1041 (this study).
Three electroporation attempts failed to delete
GSU1659 outright. This result indicates that Pcar_0202
and its orthologs lack significant histidyl-tRNA synthetase activity, and suggests that interference with
Pcar_1041 by spacer #1 would exert severe pressure on
P. carbinolicus.
The phylogenetic tree also demonstrates that the hisS
gene Pcar_1041 was not acquired laterally; it is clearly
an ancestral gene containing a proto-spacer that is not
present in its closest relatives.
Spacer #1 inhibits growth of a transgenic G.
sulfurreducens strain containing hisS of P. carbinolicus
It is not yet possible to make mutations in P. carbinolicus. Therefore, interference of spacer #1 with hisS was
tested in the more genetically tractable species
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Page 7 of 15
496
478
493
416
385
493
218
500
298
399
387
378
500
473
486
500
483
361
500
148
493
500
295
500
493
498
186
443
500
500
427
218
367
395
499
Desulfuromonas acetoxidans HisZ
Pelobacter carbinolicus HisZ Pcar_0202
Geobacter daltonii HisZ
Geobacter uraniireducens HisZ
Geobacter metallireducens HisZ
Geobacter sulfurreducens HisZ
Geobacter bemidjiensis HisZ
Pelobacter propionicus HisZ
Listeria monocytogenes HisZ
Chromatium violaceum HisZ
Nitrosococcus oceani HisZ
Pseudomonas fluorescens HisZ
Rhodoferax ferrireducens HisZ
Lactococcus lactis HisZ
Bradyrhizobium japonicum HisZ
Methanococcus jannaschii HisS
Haloarcula marismortui HisS
Thermotoga maritima HisS
Aquifex aeolicus HisS
Desulfuromonas acetoxidans HisS
Pelobacter carbinolicus HisS Pcar_1041
Pelobacter propionicus HisS
Geobacter daltonii HisS
Geobacter uraniireducens HisS
Geobacter metallireducens HisS
Geobacter sulfurreducens HisS
Geobacter bemidjiensis HisS
Chromatium violaceum HisS
Rhodoferax ferrireducens HisS
Erwinia carotovora HisS
Escherichia coli HisS
Photorhabdus luminescens HisS
Shewanella oneidensis HisS
Nitrosococcus oceani HisS
Pseudomonas fluorescens HisS
Streptococcus pneumoniae HisS
Listeria monocytogenes HisS
Bacillus subtilis HisS
Figure 4 Phylogeny of HisS and HisZ proteins. Pcar_1041 and orthologous proteins of Geobacteraceae cluster among true histidyl-tRNA
synthetases (HisS), whereas Pcar_0202 and its orthologs cluster among the HisZ proteins, which are the regulatory subunit of ATP
phosphoribosyltransferase. Confidence values are out of 500 bootstraps.
G. sulfurreducens, in which the repeat sequence of the
CRISPR2 locus (5′-GTGTTCCCCGCATGCGCGGGGATGAACCG-3′) is very similar to that of the P. carbinolicus CRISPR. A plasmid called pMA35-1 was
designed for IPTG-inducible expression of a chimeric
CRISPR construct consisting of spacer #1 of P.
carbinolicus between two copies of the G. sulfurreducens
repeat, and a transgenic strain of G. sulfurreducens
called MA159 was generated in which the native hisS
gene GSU1659 was replaced by Pcar_1041, the hisS
gene of P. carbinolicus. The tRNA-His sequences of the
two species are very different (Figure 5), suggesting that
Aklujkar and Lovley BMC Evolutionary Biology 2010, 10:230
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Page 8 of 15
P. carbinolicus tRNA-His gene
GGTGGGTGTAGCTCAGTTGGTAGA-GCACTGGATTGTGGCTCCAGTTGTCGAGGGTTCGAACCCCTTCACTCACCCCA
||||| | | |
|| |
| ||| || |||| ||| |
||| |||||| || |||
|||||||
GGTGGCTATGGTGAAGGGGTCTAACACACATGACTGTGACTCATGCATTCGTGGGTTCAAATCCCACTAGCCACCCCA
G. sulfurreducens tRNA-His gene
Figure 5
Alignment of the sequences of tRNA-His genes from P. carbinolicus and G. sulfurreducens.
the histidyl-tRNA synthetase of P. carbinolicus might
have difficulty recognizing its substrate in G. sulfurreducens. However, replacement of GSU1659 with Pcar_1041
resulted in a viable strain, which grew more slowly than
the wild type (Figure 6).
Despite prior expression of the LacI repressor protein
from plasmid pRG6 to prevent premature expression of
the chimeric CRISPR, electroporations of MA159(pRG6)
with pMA35-1 and two serendipitously obtained variants (pMA35-2 with two copies of spacer #1 and
(a)
pMA35-! with spacer #1 interrupted by a single base
pair insertion as shown in Table 1) were marginally successful (yielding zero to 4 colonies per attempt), whereas
electroporation with an equal amount of the empty vector pMA36 produced hundreds of colonies per attempt.
Electroporations of the wild type G. sulfurreducens
strain DL1, carried out in parallel, yielded hundreds of
colonies for all three chimeric CRISPR expression plasmids. These observations suggest that even leaky expression of the chimeric CRISPR containing spacer #1, or its
(b)
OD600
mM Fe(II)
100
1.00
0.10
10
0.01
0
25
50
Time (hours)
75
1
0
10
20
30
40
50
60
Time (hours)
Figure 6 Growth of G. sulfurreducens with hisS of P. carbinolicus is inhibited by spacer #1. (a) Growth on NBAF medium by reduction of
fumarate. (b) Growth on FWAFC medium by reduction of Fe(III) citrate. The strains shown are wild type G. sulfurreducens DL1 (black squares);
DL1(pMA35-2) with two copies of spacer #1 in a plasmid-borne chimeric CRISPR (white squares); transgenic strain MA159, which has hisS of P.
carbinolicus (black diamonds); MA159(pMA35-2) with both the hisS transgene and two copies of spacer #1 (white diamonds); and MA159(pMA350) with the hisS transgene and a CRISPR repeat without spacer #1 (grey diamonds).
Aklujkar and Lovley BMC Evolutionary Biology 2010, 10:230
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Spacer #1 reduces the amount of hisS RNA in transgenic
G. sulfurreducens no more than it affects hisZ RNA
Total RNA was isolated from NBAF-grown cultures of
strains containing hisS of P. carbinolicus. The amount of
hisS RNA was higher in the control MA159(pMA35-0)
strain than in the growth-inhibited MA159(pMA35-2)
strain with spacer #1 (Figure 7), and lowest in the
MA159(pMA35-!) strain that had the most severe
growth defect in a parallel growth experiment using the
same inoculum. However, when the amount of hisZ
RNA was compared across the same three strains as a
control, a similar pattern was observed (Figure 7), suggesting that reduced expression of other housekeeping
6.0 x 104
molecules per ng
mere presence as DNA, is largely incompatible with the
hisS gene of P. carbinolicus containing the matching
proto-spacer, which is present in the MA159 host, but
not with the native hisS gene of G. sulfurreducens in the
DL1 host.
Growth experiments provided further proof that
spacer #1 interferes with hisS of P. carbinolicus. The G.
sulfurreducens transformants were first checked by PCR,
restriction digestion, and sequencing to verify that the
hisS transgene and spacer #1 were intact. In one transformant, spontaneous recombination of the repeats on
either side of spacer #1, eliminating it from the chimeric
CRISPR expression plasmid, resulted in a strain called
MA159(pMA35-0) that possessed both hisS of P. carbinolicus and the repeat sequence on the plasmid, but no
spacer #1. Compared to this control that grew similarly
to MA159, the presence of spacer #1 in the other
MA159 transformants (i.e., with hisS of P. carbinolicus)
resulted in long lag periods and somewhat reduced
growth rates in NBAF medium with fumarate as the
electron acceptor (Figure 6a), and very poor growth in
FWAFC medium with Fe(III) citrate as the electron
acceptor (Figure 6b). This effect was the same with
either one copy of spacer #1 in MA159(pMA35-1) or
two copies in MA159(pMA35-2), or with a single base
pair insertion in spacer #1 in MA159(pMA35-!) - for
the sake of clarity, only MA159(pMA35-2) is shown.
The only exception was that in one experiment, triplicate cultures of MA159(pMA35-!) grew especially poorly
after three transfers in NBAF (not shown). Wild type
growth patterns were observed when any of the three
plasmids was present in the DL1 host (i.e., with hisS of
G. sulfurreducens) - for clarity, only DL1(pMA35-2) is
shown (Figure 6). Although expression of spacer #1 is
expected to be low in the absence of IPTG, growth inhibition of the MA159 strains was observed, and addition
of IPTG had no effect, indicating that expression of
chimeric CRISPR RNA was not the limiting factor for
inhibition of growth.
Page 9 of 15
MA159 MA159 MA159
pMA35-0 pMA35-! pMA35-2
MA159 MA159 MA159
pMA35-2 pMA35-0 pMA35-!
4.0 x 104
2.0 x 104
0
hisS mRNA hisZ mRNA
Figure 7 Spacer #1 has similar effects on the amounts of hisS
and hisZ RNA. The strains shown are G. sulfurreducens MA159
(pMA35-0) with the hisS transgene and a CRISPR repeat without
spacer #1 (diagonally striped bars); MA159(pMA35-2) with both the
hisS transgene and two copies of spacer #1 (speckled bars); and
MA159(pMA35-!) with the hisS transgene and a single mutated copy
of spacer #1 (diamond-patterned bars). Reverse transcription was
performed with either primer MA0329 for hisS or primer MA0442 for
hisZ, and the amount of cDNA was quantified by QRT-PCR. The
mean of three biological replicates is shown; error bars represent
the minimum and maximum.
genes besides hisS occurs when growth is slowed by the
incompatibility between spacer #1 and hisS.
The P. carbinolicus genome has fewer genes with
numerous or closely spaced histidine codons than
closely related genomes
The evidence that P. carbinolicus expresses CRISPR
spacer #1, and that spacer #1 inhibits growth of a
G. sulfurreducens strain that is dependent on hisS of
P. carbinolicus, led to the question of whether any effect
of this interference during recent evolution could be discerned in the genome of P. carbinolicus. If the expected
shortage of histidyl-tRNA were occasionally severe
enough for ribosomes to stall during translation of
genes with numerous histidine codons, one would
expect these genes to be predisposed for elimination
from the genome, because abortive expression wastes
energy and because any selective advantage of the genes
would be diminished. Missense mutations of closely
spaced histidine codons would also be favoured as long
as they did not interfere with an essential function.
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Therefore, the number of histidine codons in every gene
and the harmonic mean distance between histidine
codons in every gene were computed for P. carbinolicus
and its closest relative with a nearly completely
sequenced genome, Desulfuromonas acetoxidans
[GenBank:NZ_AAEW00000000], as well as for the completely sequenced and manually curated genome annotations of the more distantly related G. sulfurreducens
[32], G. metallireducens [33] and G. bemidjiensis (Aklujkar et al., submitted). A plot of the fraction of protein
sequences in each genome that have a given minimum
number of histidines shows that the P. carbinolicus genome is deficient in genes with 35 or more histidine
codons, and possesses none with 45 or more (Figure
8a). To identify ancestral genes that might have been
counterselected in P. carbinolicus due to close spacing
of histidine codons, an index of histidine demand was
computed as the number of histidine codons in a gene
divided by the harmonic mean distance between them.
Fewer genes with histidine demand above 5.0 are present in the P. carbinolicus genome, and none has an
index above 10.0 (Figure 8b). Despite these trends, the
overall frequency of histidine codons in the P. carbinolicus genome is 22.50 per thousand, very similar to
D. acetoxidans (23.94 per thousand), G. sulfurreducens
(20.55 per thousand), G. metallireducens (20.42 per
thousand) and G. bemidjiensis (19.76 per thousand).
This observation is consistent with the expected effect
(a)
of an acute histidyl-tRNA shortage in the vicinity of
gene transcripts with multiple or closely spaced histidine
codons undergoing translation, whereas a defect in histidine biosynthesis prior to histidyl-tRNA synthetase
would be expected to affect histidine codon usage in
general.
The P. carbinolicus genome has lost ancestral genes
with numerous or closely spaced histidines
Genes of D. acetoxidans, G. sulfurreducens, G. metallireducens and G. bemidjiensis that contain 35 or more histidine codons, or have a histidine demand index above
5.0, were examined in order to identify ancestral genes
that have reduced their histidine demand or have been
lost specifically in the P. carbinolicus genome (Additional file 1: Table S1). Many genes found in Geobacter
species are not necessarily ancestral to P. carbinolicus;
they lack homologs in either the unfinished D. acetoxidans genome or the partial genome sequences of a mixture of D. acetoxidans and D. palmitatis (D. R. L. and
coworkers, unpublished). Other genes that are present
in D. acetoxidans, but not Geobacter species, could have
been acquired after divergence from P. carbinolicus. Five
gene families actually show increased histidine demand
in P. carbinolicus compared to other Geobacteraceae,
and in many other cases, a P. carbinolicus gene has
lower histidine demand than its orthologs, but is still
above the cutoff value of 5.0, or contains a similar
(b)
1.000
Fraction of proteins with 2
histidines
Fraction of proteins
1.000
0.100
0.010
0.001
0.000
0.100
0.010
0.001
0.000
0
10
20
30
40
50
60
Minimum number of histidines
P. carbinolicus
G. metallireducens
G. bemidjiensis