Investigacin

1 YQIK
The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotein
(YqiH), a secreted !acetylmuramoyl!"!alanine amidase (YqiI), and a cytoplasmic
glycerophosphodiester phosphodiesterase (YqiK)# $e%erse transcriptase &'$ ($T!&'$) analysis
sho(ed that the yqiHIK genes are transcribed as an operon# 'onsistent (ith the in silico
prediction, (e found that the purified YqiI protein exhibited hydrolytic acti%ity to(ard
peptidoglycan sacculi# Transcription studies (ith yqiH!tre) reporter fusion strains re%ealed that
the expression of yqiHIK is sub*ected to finely tuned osmotic control, but enhanced expression
occurs only in se%erely osmotically stressed cells# &rimer extension analysis pinpointed the
osmotically responsi%e yqiHIK promoter, and site!directed mutagenesis (as employed to assess
functionally important sequences required for promoter acti%ity and osmotic control# &romoter
%ariants (ith constituti%e acti%ity (ere isolated# ) deletion analysis of the yqiHIK regulatory
region sho(ed that a +,!bp )T!rich -) segment positioned ./0 bp upstream of the !,+
sequence is critical for the acti%ity and osmotic regulation of the yqiHIK promoter# Hence, the
expression of yqiHIK is sub*ected to genetic control at a distance# 1pon the onset of gro(th of
cells of the B# subtilis (ild!type strain in high!salinity medium (.#2 3 a'l), (e obser%ed gross
morphological deformations of cells that (ere then re%ersed to a rod!shaped morphology again
(hen the cells had ad*usted to the high!salinity en%ironment# The products of the yqiHIK gene
cluster (ere not critical for reestablishing rod!shaped morphology, but the deletion of this
operon yielded a B# subtilis mutant impaired in gro(th in a defined minimal medium and at
high salinity#
4tructure and genetic organi5ation of
the yqiHIK gene cluster# ()) 6enetic
map of the yqiHIK genes in the
genome of B. subtilis# The length and
the position of the different &'$!
amplified fragments are indicated# )
bent arro( indicates the promoter
region and the direction
of yqiHIK transcription7 the position
of a putati%e factor!independent
transcriptional terminator sequence
is mar8ed by a lollipop# (B) $T!&'$!
based analysis of the
putati%e yqiHIK operon# The
amplification reactions using the
indicated -) primers (&. to &9)
(ere conducted on different
templates: c-), genomic -) as a
positi%e control, and $) as a
negati%e control# Total $) (as isolated from cells of B. subtilis strain .;/ gro(n in 433# 3,
molecular mar8er#
'ell (all hydrolase acti%ity of the purified YqiI<
4trep!tag!II protein# ("eft) 'oomassie blue!
stained 4-4!&)6= gel of the affinity purified
YqiI<4trep!tag!II protein# ($ight) >ymogram
analysis (.;) using purified B. subtilis .;/
sacculi# 4taining of the polyacrylamide gel (ith
methylene blue and destaining (ith distilled
(ater detected areas of peptidoglycan lysis#
=ight micrograms of the purified YqiI<4trep!
tag!II protein (as applied for both 4-4!&)6=
and the 5ymogram#
In silico analysis of the YqiI protein# ()) )lignment of the amino acid ())) sequences
(catalytic domain only) of the YqiI, "yt', '(l', '(l-, and Yr%? proteins from B. subtilisand
the '(l@. protein from P. polymyxa %ar# colistinus# Blac8 boxes highlight amino acids that
are in%ol%ed in the coordination of a 5inc ion in the acti%e site of amidases (.+,+9)# (B)
-omain organi5ation of B. subtilis proteins homologous to YqiI# The lengths of the proteins
are indicated# Boxes (ith hatch mar8s represent 4ec!type signal sequences7 they are present
in all amidases sho(n except for '(l'# The amidaseA, ()miA,) domain (&B0.+020) is
mar8ed# Blac8 boxes and gray triangles represent different types of cell (all!binding
domains: cell (all!binding domain 2 ('CBA2) (&B09.22) of "yt', the spore domain
(4&D$) of '(l' (&B0+0,;), the 4H,b domain of Yr%? (43002/E), and the amine domain
()3I) (&B..E9.) of '(l@# (') In silico model of YqiI based on the crystal structure of the
catalytic domain of the '(l@. protein (&-B accession number .?CF) from P.
polymyxa %ar# colistinus# The amino acids H!.+, =!2G, H!/2, and =!.9/ are predicted to
coordinate a 5inc ion in the acti%e center of the protein and contribute to en5yme acti%ity#
6ro(th properties of yqiHIK deletion
mutant strain KBB/ and strain KBB,E (lytC
cwlC cwlD yrvJ yqiI)# () and B) 'ultures
of B. subtilis (ild!type strain .;/ (blac8
circles), its H(yqiHIK::neo) deri%ati%e strain
KBB/ ((hite circles), and the lytC cwlC
cwlD yrvJ yqiI quintuple mutant strain
KBB,E (gray circles) (ere gro(n in 433
alone ()) or in 433 containing .#2 3 a'l
(B)# (') The morphologies of B.
subtilis strains .;/, KBB/, and KBB,E (ere
analy5ed by phase!contrast microscopy at
different time points (indicated by arro(s)
during the gro(th of the cultures# 4cale bar,
+ Im#
Bluorescence and scanning electron
microscopy of salt!stressed B. subtilis cells#
()) 'ells of (ild!type strain .;/ and its
mutant deri%ati%es KBB/ (yqiHIK) and
KBB,E (lytC cwlC cwlD yrvJ yqiI) (ere
gro(n in 433 (ith .#2 3 a'l for .2 h (time
point 9) (Big# 9B) and obser%ed by both
phase!contrast and fluorescence (after
staining (ith the "i%eJ-ead Bac"ight
bacterial %iability 8it) microscopy# 4cale bar,
+ Im# (B and ') 'ultures of strain .;/ (ere
gro(n to the early exponential phase
(D-+E/ of 0#E to 0#G) in 433 alone (B) or in
433 containing .#2 3 a'l (') and (ere
obser%ed by scanning electron microscopy# The magnification (as set at K/,000, and the
scale bar represents 9 Im#
Influence of the )T!rich region
located upstream of
the yqiHIK promoter region on
osmotic induction of yqiH-
treA reporter gene expression# ())
-) sequence of the ,00!bp
fragment fused to the
promoterless treA reporter gene in
strain KBB.+# The )T!rich region is
highlighted by a blac8 box# The -)
fragment sho(n is defined as H0#
)rro(s indicate three different
truncations (H. to H,) of the ,00!bp
region originally used to construct
the yqiH-treA gene fusion# The positions of the L,+ and L.0 yqiHIK promoter regions, the
transcriptional initiation site, the ribosome!binding site of the yqiH gene, and its
translational start codon of the yqiH reading frame are indicated# (B) &romoter acti%ity and
osmoregulation of reporter strains carrying the %arious deletion constructs in cells
culti%ated either in 433 or in 433 containing .#2 3 a'l#
Influence of the )T!rich region located
upstream of the yqiHIK promoter region
on osmotic induction of yqiH-
treA reporter gene expression# ()) -)
sequence of the ,00!bp fragment fused
to the promoterless treA reporter gene in
strain KBB.+# The )T!rich region is
highlighted by a blac8 box# The -)
fragment sho(n is defined as H0# )rro(s
indicate three different truncations (H.
to H,) of the ,00!bp region originally
used to construct the yqiH-treA gene
fusion# The positions of the L,+ and L.0 yqiHIK promoter regions, the transcriptional
initiation site, the ribosome!binding site of the yqiH gene, and its translational start codon
of the yqiH reading frame are indicated# (B) &romoter acti%ity and osmoregulation of
reporter strains carrying the %arious deletion constructs in cells culti%ated either in 433 or
in 433 containing .#2 3 a'l#
Dsmotic regulation of yqiH-
treA expression and identification of
the yqiHIK transcription start site by
primer extension analysis# ())
-etermination of Tre) acti%ity in cells
of strain KBB.+ (yqiH-treA) cultured
in 433 (ith increasing a'l
concentrations# )ll cell samples (ere
har%ested for Tre) en5yme acti%ity
measurements at the mid!exponential
gro(th phase (D-+E/ of about .#0 to
.#+)# (B) &rimer extension analysis of
the yqiHIK transcript in cells of B.
subtilis strain .;/ carrying plasmid
pKB.; (yqiHM) cultured either in
433 (L) or in 433 (ith .#2 3 a'l
(N)# (') ucleotide sequence of
the yqiHIK promoter region# The
transcription initiation site (N.) is
indicated by a bent arro(# The L,+
and L.0 regions are highlighted (ith
gray boxes and are separated by .E bp#
The ribosome!binding site ($B4) of
the yqiH gene, its start codon, and the
!terminal amino acid sequence of
the YqiH protein are sho(n#
2 YQIK HYPOTHETICAL PROTEIN [ ESCHERICHIA COLI STR. K-12
SUBSTR. W3110 ]
2.1 SUMMARY
Gene symbol
yqiK
Gene descri!ion hypothetical protein
Loc"s !#$ Y75_p2977
Gene !ye
Protein coding
Re%&e' s!#!"s PROVISIONA
Or$#nism !"cherichia coli "tr# K$%2 "&'"tr# ()%%* +"train, K$
%2- "&'"train, ()%%*- old$na.e, !"cherichia coli
"tr# K%2 "&'"tr# ()%%*/
Line#$e 0acteria1 Proteo'acteria1 2a..aproteo'acteria1
!ntero'acteriale"1 !ntero'acteriaceae1
!"cherichia
2.2 GENOMIC CONTEXT
&e'"ence( N3_**7779#% +)%9%52*##)%9)%4%/
2.3 GENOMIC REGIONS, TRANSCRIPTS, AND PRODUCTS
Genomic &e'"ence( N3_**7779
2)* +I+LIOGRAPHY
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