Heat and DNA damage induction of the LexA-

like regulator HdiR from Lactococcus lactis is

mediated by RecA and ClpP

By

Jain Amey Manojkumar

2017A1PS0864G

Under the supervision of:

Prof. MALABIKA BISWAS
Department of BIOLOGICAL SCIENCE,
BITS Pilani, K.K Birla Goa Campus
Submitted in partial fulfilment of the requirements of
BIO F266-
STUDY PROJECT
 1. INTRODUCTION
The SOS response is a typical example of bacterial cell response to damaged DNA.
LexA is a homologue of SOS regulator. It is absent in some bacteria, example-
Lactococcus Lactis. A negative transcriptional regulator HdiR were indentified is
these organism. They induce target gene expression both upon DNA damage and
heat shock. HdiR is able to carry out a self-cleavage reaction in vitro at high pHs,
while in vivo it undergoes RecA-dependent self-cleavage in the presence of a
DNA-damaging agent. Intriguingly, the N-terminal cleavage product of HdiR
retains DNA binding activity, and only when degraded by the Clp protease, is gene
expression induced. Thus, the activity of HdiR in response to DNA damage is
controlled by sequential proteolysis, involving self-cleavage and Clp-dependent
degradation of HdiR.

All living organisms have the ability to repair damaged DNA. However, in the
process elimination may occur. The current paradigm of the SOS response has
been established by Extensive studies of the Gram-negative bacterium Escherichia
Coli. Under physiological conditions, the SOS regulon is repressed by the LexA
bound to its consensus binding sites located in the promoter regions of target genes
including lexA itself, recA and the umuDC operon.

Upon DNA damage, RecA binds to single stranded DNA and forms nucleoprotein
filament (together denoted as RecA*), which possesses recombinase and co-
protease. Self-cleavage occurs between the Ala84 and Gly85 residues of LexA
resulting in inactivation of the repressor activity and induction of the SOS regulon.
Because the activity of the PolV is mutagenic, elaborate mechanisms are employed
to keep the level of the Umu proteins to a minimum. At the transcriptional level,
expression of the umuDC operon is tightly regulated by the LexA, while at the
post-translational level the amount of the mutagenically inactive and active forms
of the UmuD are controlled by the Lon and ClpXP protease.

In organisms other than E. coli , the induction and regulation of the SOS response
pathways are less well understood. SOS-like responses have been detected in
several bacteria and the LexA homologues characterized to date appear to be
functionally conserved. In the present study, a novel LexA like repressor has been
identified, HdiR, from the Gram-positive bacterium, Lactococcus lactis that co-
operates the response to both DNA damage and heat shock in a single regulatory
molecule.
 2. APPROACH
L. lactis strains were grown in M17 medium at 300 C supplemented with 0.5%
glucose. When needed, chloramphenicol (6 mg ml-1) or tetracycline was added in
growth media. E. coli strains were cultivated at 370 C in Luria–Bertani (LB)
medium supplemented with ampicillin (50–100 mg ml-1) and/or kanamycin (25
mg ml-1).

A replacement combination technique was used to construct the mutant strain

carrying a 64 bp in-frame deletion in the hdiR gene to express HdiR without the
putative HTH motif. For this purpose, PCR products generated with primers
p15/p16 and p17/p18, respectively, were digested with XbaI/PstI and PstI/SalI,
respectively, and ligated with XbaI/SalI cut pGhost8. The resultant plasmid,
encoding two additinal ALA residues at the deletion site in hdiR was introduced
into MG1363 followed by plasmid integration and excision. The mutant strain
carrying an in-frame deletion in hdiR was assigned as PV114.

It was complemented by cloning PCR amplified HdiR gene region. The resulting
plasmid pKS47 was used to transform MG1363 and PV114 to obtain KS73 and
KS74 respectively. An overexpression system for the HdiR in MG1363 and mutant
derivatives was created. Molecular cloning techniques were performed essentially.
Identification of the hdiR gene from MG1363 was done and Sequence comparisons
were accomplished using the National Center for Biotechnology Info. (NCBI)

RNA extraction and Northern blotting analyses: Total RNA was isolated from L.
lactis strains grown exponentially at 25C or 30C to an optical density OD600 =
0.3–0.4, where they were either heat stressed by transferring from 25C to 38.5C, or
kept at 30C and treated with 3 mM MMC. Cell samples were withdrawn at the
time intervals of 0, 5, 20 or 45 min when incubated with MMC. RNA isolation,
blotting and hybridization with radiolabeled probes were carried out. The DNA
probe for clpP and RecA genes from MG1363 with umuC gene were generated br
PCR using primer pairs p11/p12, p7/p8, p9/p10 .RNA amounts on the membrane
were corrected by probing the membranes with a probe specific for L. lactis 16S
rRNA.

Complementation of the PV114 strain with pKS47 was analysed using the
Bioscreen C-monitoring system. Shortly, PV114 and MG1363 containing the
pCI372 (control strains) and the KS74 (PV114/pKS47) and the KS73 (MG1363/
pKS47) were cultivated at 300 C and at 370 C as described above.
Overexpression and purification of the HdiR was also done. Coding region of
HdiR was amplified using primer p21/p22 followed by cloning in respective sites.
Purified His6-Hdir from E.coli was used for custom antibody production in rabbit
and DNA gel mobility shift experiment.

Gel Mobility Shift: DNA fragments corresponding to the putative promoter regions
of the hdiR (206 bp) from MG1363, umuC (252 bp) and recA (155 bp) were
generated by PCR using primer pairs p23/p24, p26/p27 and p28/p29 respectively.
The hdiR upstream region containing the IR2, but not the IR1, was amplified using
primers p15 and p25. Oligonucleotide pairs were annealed. The effect of
autocleavage on DNA binding was studied using His6-HdiR (12.5 ng and 25 ng)
that had been incubated in 50 mM Tris- HCl (pH 7) or 50 mM glycine (pH 10) at
room temperature for 16 h. Gel-shift reactions were incubated at 25∞C for 15 min
followed by electrophoresis on a 5% polyacrylamide gel. Gels were stained with
ethidium bromide, scanned with a Fluor-S imager and analysed using
QuantityOne-software (Bio-Rad).

HdiR self-cleavage analysis: Self-cleavage of the His6-HdiR was studied in the

pH range of 6.0–10 using a buffer system containing either 50 mM Bis-Tris, Tris-
HCl or glycine. The reaction products were separated in a 4–12% SDS–PAGE
(Invitrogen) followed by staining with Coomassie Brilliant blue R-250, or transfer
to a nitrocellulose membrane (0.45mm, Bio-Rad) for Western blots with His6-
HdiR antibodies (1:5000) and HRP-conjugated goat anti-rabbit IgG (Bio-Rad)
1:50000. Detection was performed using the SuperSignal West Dura Extended
Duration kit according to Pierce. Membranes were scanned on a GS-525 Molecular
Imager System (MultiAnalyst, Bio-Rad).

The components from the His6-HdiR self-cleavage reaction were separated by

reversed phase chromatography on a 1.0 \ 20 mm C1 column using a linear
gradient (0–100% in 60 min) of acetonitrile in 0.1% trifluoroacetic acid. The peaks
were automatically collected on a SMART™ (Pharmacia) liquid chromatograph
and subjected to N-terminal sequence nalysis using a Procise 494 A sequencer
(Perkin-Elmer).

Determination of HdiR stability in vivo: Stability of the HdiR in the presence of

MMC was studied by cultivating L. lactis strains at 300 C to OD600 = 0.5 followed
by the addition of 3 mM MMC and CAM (50 mg ml-1) after 1 min to block the
protein synthesis. Samples were with-drawn at indicated time points and Protease
Inhibitor Cocktail was added prior to centrifugation. HdiR stability under heat
stress conditions was studied by shifting exponentially growing (OD600 = 0.5)
KS78, KS79 and KS80 cells from 250 C to 38.50 C. Protein synthesis was blocked
after 5 or 15 min by CAM and samples (15 ml) were withdrawn at indicated times.
Cell free extracts were obtained after homogenizing the cells with glass beads and
centrifugation. Protein concentrations were determined with the Protein Assay kit
(Bio- Rad). An equal amount of the protein (50 mg) was separated in a SDS–
PAGE (12%) (Invitrogen) and the HdiR stability was analysed by Western
blotting.

3. Results

Firstly, in search of substrate proteins for the L. lactis Clp protease, we examined
whether the L. lactis ssp. Cremoris MG1363 genome encodes a homologue of
UmuD; which turns out to be ‘NO’. Furthermore, a search of the L. lactis ssp.
Lactis IL1403 genome sequence failed to reveal an umuD gene, although genetic
evidence has established that UmuC is present in L. lactis IL1403. However, when
we did the search using an UmuD-like protein encoded by the Tn5252 transposon
element present in the Streptococcus pneumoniae genome, we retrieved a protein
with 32% identity. The region of the highest similarity is centred on the three
conserved domains typical of the LexA-family of proteins, including LexA,
UmuD, MucA, RumA and cI-like phage repressors. The amino acids Ala84,
Gly85, Ser119 and Lys156 involved in the RecA dependent self-cleavage of the E.
coli LexA are also conserved in the IL1403 YnaB. Distinct from the transposon
UmuD, YnaB carries a putative N-terminal helix–turn–helix motif and is therefore
classified as a putative transcription regulator. Thus, based on the sequence
analysis YnaB has resemblance to both LexA and UmuD. Notably, a BLAST
search against the IL1403 genome suggests that L. lactis is devoid of conserved
LexA.

Further, we sequenced the gene encoding the YnaB orthologue from MG1363,
and it was named as a heat shock and DNA-damage induced regulator (hdiR). The
hdiR gene encodes 252-aminoacid residues, which shares 90% identity with the
YnaB from IL1403 and homologues are present in species of Streptococcus and
Staphylococcus(26–32% overall amino acid identity). In L. lactis MG1363, the
hdiR gene is preceded by a ribosome binding site as well as two inverted repeats.
The more distant inverted repeat 2 (IR2) resembles a typical rho-independent type
transcription terminator. The proximal inverted repeat 1 (IR1) is located two
nucleotides upstream of the putative -35 region. No apparent transcription
terminator structures were identified immediately downstream of hdiR, suggesting
that hdiR forms a dicistronic operon together with the downstream located ynaA
gene. Hence, HdiR expression is regulated by heat and DNA damage in L. lactis
MG1363. In E. coli, autoregulated expression of LexA is induced by DNA
damaging conditions. Because HdiR resembles LexA, we examined whether the
DNA crosslinking agent, mitomycin C (MMC), induces HdiR expression.
Furthermore, hdiR expression in MG1363 was induced ~15-fold by 20 min
exposure to MMC. Also, If hdiR is induced by other environmental conditions,
such as heat shock, cold shock at or low pH, only heat-shock affected hdiR
expression.

Curiously, prolonged incubation at an elevated temperature showed that hdiR

expression was re-repressed back to the level prior to induction already after 20
min. In the presence of MMC, however, the level of hdiR expression was still six
times the uninduced level after 60 min. HdiR is a DNA-binding protein that
regulates its own expression.

We constructed a mutant strain (PV114) that expresses a derivative of HdiR

lacking the HTH motif potentially involved in DNA binding. Northern blot
analysis revealed that in the absence of DNA damage, the expression was
increased 20-fold compared with the level in wild-type cells, and it was unaffected
by DNA damage . As these results suggested that hdiR expression is auto
regulated, we used a mobility shift assay to examine if HdiR is able to bind to its
promoter region. To this end, we purified HdiR from E. coli cells expressing a
histidine tagged derivative of HdiR. DNA fragment covering -221 to +5 relative to
start codon shows that increasing amounts of HdiR retarded the mobility of the
fragment whereas the fragment covering from -372 to -82 relative to translational
start site, binding was abolished. Hence, we could establish that IR1 contains the
target sequence of HdiR.

We were unable to detect any changes in the overall protein patterns between the
wild type and the PV114 mutant strains when cells were grown in a chemically
defined media at 300 C. Alternatively, we searched the IL1403 genome sequence
with the IR1 core motif and found an almost identical sequence that partially
overlaps the putative -10 region of the umuC gene. Southern analysis of MG1363
with a probe derived from the IL1403 umuC gene indicated that MG1363 is devoid
of an umuC homologue. Therefore, we examined if HdiR binds to the umuC
promoter region amplified from the IL1403 genome. In gel retardation
experiments, HdiR, indeed, retarded fragment carrying the IL1403 umuC
promoter. To confirm that HdiR regulates umuC expression in vivo, we introduced
a plasmid carrying the IL1403 umuC structural gene and its putative promoter
region (pKS50) into wild type (MG1363) and PV114 cells to obtain PV121 and
PV122 respectively. When PV121 and PV122 were treated with MMC, Northern
blot analysis revealed two umuC-specific transcripts, which both were induced by
MMC in wild type cells (PV121) and only marginally in the strain encoding HdiR¢
(PV122) (Fig. 2C). However, in the absence of MMC, the expression of both
umuC transcripts were 4.4-fold higher in PV122 compared with PV121, showing
that HdiR regulates the expression of umuC from IL1403.

In many bacteria, LexA regulates the expression of recA. Therefore, we

investigated if HdiR binds to the recA promoter region of IL1403. However, we
were unable to detect such a binding. Furthermore, Northern blot analysis showed
that expression of recA was identical in MG1363 and PV114 indicating that HdiR
is not a repressor of recA expression.

Cleavage of HdiR is stimulated by RecA:

LexA protein family has the ability to perform self-cleavage in vivo requiring an
activated form of RecA, while self-cleavage occurs spontaneously in vitro at high
pH . To investigate if HdiR undergoes pH-dependent self-cleavage, we incubated
the purified His6-HdiR protein at various pHs and observed that self-cleavage of
His6-HdiR proceeded spontaneously at pH 8.0–10, resulting in two products of ~14
and ~16 kDa respectively. After raising polyclonal antibodies against His6-HdiR
protein, we analysed the cleavage products by Western blot analysis and did not
detect any additional protein bands . N-terminal sequence analysis of the smaller
C-terminal cleavage product revealed that the cleavage had occurred between the
Ala126 and Gly127 residues.

Next, we examined if RecA stimulates HdiR self-cleavage in vivo. However, the

chromosomally expressed HdiR was not detectable by Western blot analysis, either
in the presence or absence of heat or MMC. Therefore, we facilitated the detection
by overexpressing HdiR from a plasmid carrying the hdiR gene inserted behind a
constitutive promoter. The resulting plasmid, pKS49, was transferred into wild
type and the recA mutant VEL1122 to obtain the strains KS78 and KS80
respectively. Subsequently, we examined the stability of HdiR under DNA
damaging conditions by exposing both strains to MMC followed by inhibition of
protein synthesis by chloramphenicol. At the time points indicated, protein extracts
were analysed by Western blot analysis. The result revealed that in the strain
lacking RecA (KS80), the amount of HdiR remained unaltered 45 min after the
addition of CAM, whereas HdiR was rapidly cleaved in the presence of RecA,
leaving essentially no unprocessed HdiR 30 min after blocking the protein
synthesis. These results show that the self cleavage of HdiR in vivo is dependent
on the RecA.

When the in vivo stability of HdiR was analysed , we found that the amount of
intact HdiR in the wild type (KS78) cells decreased, a little slowly, after the
addition of CAM, whereas HdiR remained stable in the recA mutant cells.
Furthermore, we were unable to detect the cleavage products in recA mutant cells,
whereas a small amount of fragment appeared in samples obtained from the wild
type cells, indicating that self-cleavage of HdiR occurs also under heat-shock
conditions, butonly low level. Now, we wished to determine if recA plays a role in
inducing hdiR expression under these conditions. When we examined hdiR
expression in the recA mutant cells, we found, as expected, that induction by
MMC was eliminated in the absence of RecA. We also observed that induction by
heat was completely abolished in cells lacking RecA, demonstrating that RecA,
indeed, is required for heat induction of hdiR.

ClpP modulates HdiR activities:

In accordance to many Northern analysis, the maximum induction level of hdiR

expression is reached between 15 and 30 min after addition of MMC. While
comparing the slow induction of hdiR expression in response to MMC with the
rapid self-cleavage of overexpressed HdiR under the same conditions, the results
suggested that cleavage of HdiR might not be sufficient to induce expression.
Therefore, we examined the DNA binding activity of self-cleaved HdiR.
Surprisingly, HdiR incubated overnight at pH 10 was still capable of retarding the
hdiR and umuC promoter fragments in the gel-shift assay, where the migration of
the DNA–protein complex was faster compared with the reactions containing equal
amounts of HdiR incubated at pH 7. These results show that HdiR cleavage
products are still able to bind the target sequence.

When observing self-cleavage of HdiR in vivo, we had noted that only the 14.5
kDa N-terminal cleavage product was visible and that this product also appeared
unstable. Given the resemblance of HdiR to LexA and UmuD, which both, after
processing, are targets of the Clp proteolytic complex, we examined HdiR stability
in a strain lacking ClpP. After MMC addition, we found that the half-life of
unprocessed HdiR was increased ~30% in the absence of ClpP compared with the
wild type cells. Furthermore, in the clpP mutant, both of the HdiR cleavage
products were visible, and while the half-life of the 14.5 kDa fragment in the wild
type cells was 22 min ± 2min (between 15 and 60 min), this fragment was not
degraded in the clpP mutant cells (Fig. 5B). Hence, both of the HdiR cleavage
products are targets of the Clp protease.

Next, we studied the effect of ClpP on HdiR stability after heat shock and saw that
while the amount of the HdiR decreased in the wild type cells, no significant
change was detected in cells lacking ClpP. Additionally, a faint band at 14 kDa
could be detected in all the time points of the wt cells, while in the clpP mutant
strain it was only detected in the sample withdrawn 45 min after the addition of
CAM.

In order to confirm that ClpP modulates the activity of the HdiR in vivo, hdiR
expression by Northern analysis of the wt and clpP mutant strains was analysed.
The result demonstrates that in the absence of ClpP the induction of the hdiR
expression in response to MMC and heat was reduced fourfold compared with the
induction obtained in the wt cells. Thus, cells lacking ClpP are restricted in their
ability to induce the HdiR regulon demonstrating that ClpP modulates its activity
in vivo.

HdiR is required for optimal growth of MG1363:

Data demonstrates that HdiR activity is modulated by heat and MMC. Now, we
wish to determine if HdiR is important for viability under these conditions.
Therefore, we attempted to construct a deletion mutant lacking the entire hdiR
reading frame. However, our repeated attempts for creating such a strain have been
unsuccessful. Alternatively, the ability of the wt and PV114 mutant cells to grow
in the presence and absence of MMC and at an elevated temperature (370 C) were
examined, and no difference was found in the colony forming ability between the
two strains. However, PV114 generally required longer time of incubation to form
colonies of same size as MG1363 under all conditions. PV114 was complemented
with a plasmid (pKS47) encoding HdiR, but not the downstream encoded YnaA, to
obtain KS74. pKS47 was also introduced into MG1363 yielding KS73. The growth
rates of the KS73, KS74 and their parental strains (MG1363, PV114) carrying the
vector, pCI372, were measured using the Bio-screen monitoring system.
The results from the growth experiments carried out at 300 C without MMC using
five parallel samples revealed that while the doubling time for MG1363(pCI372)
was 59.1 min (SD 1.66), it was increased in PV114(pCI372) to 78.2 min (SD
2.26). When the strains were cultivated at an elevated temperature (300 C), the
PV114(pCI372) failed to initiate growth, whereas the KS74 and KS73 and
MG1363(pCI372) grew exponentially with the generation times 149.8 min (SD
10.91), 138.2 min (SD 4.97) and 152.8 min (SD 4.57) respectively. These results
show that when HdiR lacks the HTH domain, the strain has a growth defect, which
abolishes growth at a high temperature under the conditions used.

4. Discussion
 In bacterial cells, DNA damage response relies on LexA like transcriptional
regulators, which in the absence of DNA damage bind to DNA sequences
located upstream of target genes, and upon DNA damage undergo RecA
mediated self-cleavage to relieve the repression of target gene expression.

 We have characterized a transcriptional regulator, HdiR, from the Gram-

positive bacterium, Lactococcus lactis that possesses several LexA like
features including negative auto-regulation of its own synthesis, induction
by MMC and self-cleavage between the conserved Ala and Gly residues.

 Prior to LexA-like selfcleavage, RecA binds to single-stranded DNA

regions, while polymerizing into a nucleoprotein filament. This change
enables RecA to act as co-protease in the self-cleavage reactions of LexA,
UmuD and phage repressor proteins. Thus, a similar activation of RecA is
likely to precede the HdiR self-cleavage.

 When the stability of the overexpressed HdiR was assessed in wt cells

exposed to MMC, only the larger, N terminal part of the two HdiR cleavage
products was visible and the half-life of this fragment was ~22 min.

 However, in the absence of clpP encoding the proteolytic subunit of the Clp
protease complex, both of the cleavage products were stabilized suggesting
they are targets of this protease.

 We also noted that the clpP mutation essentially abolished the induction of
hdiR expression by MMC, indicating that the N-terminal HdiR cleavage
product carrying the HTH motif retained its DNA binding ability. This
notion was confirmed by gel retardation studies, where the cleaved HdiR
retarded both the hdiR and the umuC promoter fragments.
 The Clp proteolytic complex has previously been implicated in the
degradation of self-cleavage products. The UmuD¢ is rapidly degraded by
the ClpXP to avoid excess mutagenesis and in very recent studies also the
products of self-cleaved LexA are shown to be subtrates for the ClpXP
protease. Thus, the Clp proteolytic complex appears to play a conserved role
in turnover of self-cleaved protein products.

 In the case of HdiR, the Clp proteolytic complex regulates the DNA damage
response, as the N-terminal cleavage product can still repress gene
expression, and only when degraded by ClpP, is the response to DNA
damage induced. The deleterious effect of accumulation of the LexA DNA-
binding domain to cell survival after DNA damage suggests a similar
regulatory mechanism in E. coli.

 In addition to MMC, hdir expression is also induced by heat. Although HdiR

was unstable (half-life ~20 min) under these conditions, it was threefold more
stable than in the presence of MMC. However, an ~15-fold induction of hdiR
expression was achieved already ~5 min after applying of heat-shock, whereas
20 min were needed to reach the same level when cells were stressed with
MMC, suggesting that the stability of HdiR does not play a crucial role in de-
repressing the HdiR regulon under heat-stress.

 However, induction of hdiR expression by heat was completely eliminated

in the recA mutant cells and was greatly reduced in the absence of clpP,
demonstrating that both RecA and ClpP are clearly important for the heat-
shock induction. Although additional proteins may be involved in this
regulation, our data show that common elements are involved in both the
DNA damaged induced and the heat-mediated induction of the HdiR
regulon.

 LexA homologues are present in a number of bacteria, particularly in those

with larger genomes. Curiously, Staphylococci contain both HdiR and LexA
homologues, suggesting that the two proteins have separate biological
functions. However, Lactococcus and Streptococcus species appear to be
devoid of LexA and, thus, HdiR could be the DNA damage response
regulator in these organisms. To address this issue, we made several
attempts to identify HdiR target genes; however, we only identified hdiR in
L. lactis MG1363, while umuC is a target for HdiR in L. lactis IL1403.
 In addition, we specifically examined if recA expression is repressed by
HdiR, but did not detect any differences in the expression between the wt
and PV114 mutant cells expressing HdiR¢. Based on a recent study, we also
examined if HdiR is a positive regulator of recA expression.

 After exposing wt and hdiR mutant cells to MMC, we found that recA was
only slightly induced (~twofold), and that this induction was slightly
reduced in cells carrying HdiR¢ lacking the HTH domain. Thus, HdiR has
only a marginal effect on recA expression.

 When we examined the phenotype of the mutant cells expressing HdiR¢, we

found that they were as resistant as the wt cells to MMC, but were unable to
grow at high temperature. Therefore, the timely expression of a gene
controlled by HdiR appears to be required for growth at high temperatures.

 In this paper, we have shown that the N-terminal half of HdiR remains
active following self-cleavage. Interestingly, the C-terminal half of the
protein was found to be highly unstable and only detected in cells lacking
ClpP, indicating that it might also harbour an important biological function.

 In our study, we have identified a novel type transcriptional regulator whose

expression responds to both DNA damage and heat-stress. A link between the
response to heat shock and DNA damage in L. lactis has previously been
suggested by the findings that disruption of the L. lactis recA gene results in
temperature sensitivity, and this sensitivity is suppressed by disruption of a
gene, trmA, which also suppresses the temperature sensitivity of a clpP
mutation. Our present findings suggest that HdiR may be such a link.

5. Ackowledgement

I would like to thank Prof. MALABIKA BISWAS for guiding me in this

endeavor.

6. REFERENCE
http://www.blackwellpublishing.com/products/journals/suppmat/mmi/mmi3713/m
mi3713sm.htm

© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 609–621

http://www.ncbi.nlm.nih.gov/BLAST