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Journal of Bacteriology, June 2004, p . 4025-4029, Vol . 186, No . 12
The
Last Gene of the fla/che Operon in Bacillus subtilis, ylxL,
Is Required for Maximal
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| ABSTRACT |
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ylxL was found to be the last gene of the fla/che operon in
Bacillus subtilis and is cotranscribed with the gene for the
flagellum-specific alternate sigma factor,
D .
The ylxL gene was disrupted by insertional mutagenesis, and
the resultant mutant strain was found to be compromised for
D-dependent
functions .
| TEXT |
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In Bacillus subtilis, the structural genes that encode the hook-basal
body complex (HBB), several genes controlling chemotaxis, and
the gene for the alternate sigma factor,
D,
are found adjacent to one another in a 26-kb region of the bacterial
chromosome called the fla/che region (15) .
Several studies have demonstrated that these genes comprise a single
operon (2, 10, 16,
18), while genes encoding these functions in the
enteric bacterium are found in at least seven operons located
throughout the bacterial chromosome (7) . Many of
the genes within the fla/che operon have been cloned,
sequenced, and characterized (1, 15);
however, the 3' end of the operon has yet to be mapped, and the
function of the ylxL gene product has not been analyzed .
Genetic organization of ylxL. An open reading frame,
originally referred to as orfC and later renamed ylxL
as a result of the B . subtilis genome project (3),
was identified immediately downstream of the structural gene
for
D,
sigD (Fig . 1) . While sigD, located 26 kb from
the major promoter for this operon, has been shown to be part of
fla/che, (2, 16), it was not
known if ylxL is part of the fla/che transcription
unit . Previous studies demonstrated that insertions between sigD
and ylxL resulted in defects in chemotaxis (9,
18), swimming (9), and motility
(10) . Additionally, sequence analysis of this
intergenic region failed to identify a transcriptional terminator,
leading to the speculation that ylxL is part of the fla/che
operon . Furthermore, the rpsB gene immediately downstream of
ylxL is known to encode a ribosomal protein (5)
and appears to be monocistronic with its own promoter (http://genolist.pasteur.fr/SubtiList),
suggesting that ylxL may be the final gene of the fla/che
operon .
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RPA of ylxL. To determine whether ylxL is the
last gene of the fla/che operon, RNase protection assays (RPA)
were performed using the RPA II kit from Ambion . Riboprobes were
synthesized for this analysis by first cloning the intergenic regions
highlighted in Fig . 1 into plasmid pGEM-Zf7(+) from
Stratagene . These intergenic regions were amplified by PCR and cloned
into the plasmid in the orientation that allows for production of
antisense RNA in an in vitro transcription reaction . T7 RNA
polymerase and [
-32P]UTP
were used in such a reaction to yield body-labeled riboprobes .
Full-length protection of the riboprobes generated from plasmids pHW1
and pHW2 was obtained (data not shown), confirming that sigD
is part of the fla/che operon and demonstrating that ylxL
is cotranscribed with sigD . The data obtained using the
riboprobe generated from pHW3 were less straightforward .
The 416-base riboprobes synthesized from pHW3 include nonhomologous sequence from the vector and 368 bases of sequence complementary to the region spanning the ylxL and rpsB genes (Fig. 1) . These riboprobes were used to identify the 3' end of the fla/che operon . The rpsB gene encodes the 30S ribosomal protein S2 (5) and is not considered to be part of the fla/che operon . Total RNA was extracted from wild-type B . subtilis cells (LMB7) grown in 2XSG sporulation medium and harvested at T0 and T0.5, when sigD expression is known to be maximal (13) . Riboprobes were incubated with isolated RNA to allow annealing of complementary strands . Single-stranded portions of the riboprobes were digested with RNase provided with the RPA II kit, while regions of riboprobes complexed in the RNA duplex with ylxL-rpsB mRNA were protected from digestion and resolved by electrophoresis on a 5% denaturing gel . The resulting autoradiograph of the dried gel showed three primary products of 368, 190, and 119 nucleotides (Fig. 2A) .
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The 368-base fragment is consistent with the lack of transcription
termination by RNA polymerase at the end of ylxL and read-through
into the rpsB gene: this is the predicted size for protection
of homologous sequences contained within the riboprobes synthesized
from pHW3 . The significantly more abundant 119-base fragment is
of the expected length for rpsB transcripts initiating at its
own promoter, according to the SubtiList website (http://genolist.pasteur.fr/SubtiList) .
The protected 190-base fragment, however, suggests the termination
of transcription at the end of the ylxL coding sequence prior
to this promoter . In order to confirm the last two assignments,
an end-labeled probe of the pHW3 region used for riboprobe synthesis
was generated . The appropriate region of pHW3 was amplified by PCR
and 5' end labeled in a kinase reaction using [
-32P]dATP .
The resulting DNA probe was incubated with B . subtilis RNA and
subjected to S1 nuclease digestion . As with the riboprobe assay,
protection of the DNA probe from digestion requires that it
anneal to complementary mRNA emanating from the ylxL-rpsB
intergenic region, but it would be detected by autoradiography only
if the 5' end of the DNA probe were found within the duplex . While
the 368- and 119-base fragments were still detected following
S1 digestion, the 190-base fragment was no longer evident (Fig .
2B) . These results demonstrate that the 5' end of the probe
is not found within the 190-base protected fragment, and this
is likely due to protection of the 3' end of the probe by ylxL
mRNA . Taken together, the data obtained from the RPA show that
ylxL is the last gene of the fla/che operon, although some
read-through into the downstream rpsB gene is evident .
Studies of ylxL function. ylxL, a 501-bp gene
found at the 3' end of the fla/che operon in this study,
encodes a protein of unknown function . BLAST analysis showed that the
predicted protein product of the ylxL gene has low similarity
to other proteins, also of unknown function . However, due to previous
genetic studies of the ylxL region (9,
18) and its determined location within the fla/che
operon, we postulated that it might play a role in flagellar and
chemotaxis functions . To study its predicted function, an insertional
disruption of ylxL was generated by transformation with the
disruptional plasmid pHW4 into the wild-type strain LMB7, yielding
LMB231 (Table 1) . pHW4 contains a 169-bp insert
complementary to the 5' end of the ylxL open reading frame
immediately downstream of its translational start codon . Insertion of
this plasmid disrupts ylxL without affecting expression of
sigD as determined by anti-D
immunoblot analysis, described previously (16).
D
protein levels in the ylxL mutant LMB231 were found to be
comparable to those found in the wild-type strain (Fig .
3A) and were undetectable in the
D
null mutant, LMB10 . Furthermore, the phenotype of the ylxL
mutant was found to be the result of the insertional disruption in
the ylxL gene and not to an unlikely polar effect on rpsB
expression . Insertion of the identical plasmid sequences that
are found in the ylxL disruption between ylxL and rpsB in
strain LMB281 did not produce the mutant ylxL phenotype (data
not shown) .
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Flagellin expression. To determine the biological importance of
ylxL in flagellin gene expression, strain LMB237 bearing both
the ylxL disruption and a hag-lacZ translational fusion
(Table 1) was grown in 2XSG sporulation medium, and
ß-galactosidase activity was measured as described previously (13) .
Under these conditions, flagellin gene expression increases
throughout exponential growth and peaks shortly thereafter (13),
as found for hag-lacZ expression in the wild-type strain,
LMB25 (Fig . 3B) . However, the reporter strain
bearing the ylxL mutation, LMB237, displayed a drastic
decrease in ß-galactosidase activity (Fig . 3B) . In
this strain, hag-lacZ expression ranged from 50 to 100 Miller
units throughout growth, indicating that ylxL is required for
high-level flagellin gene expression that is developmentally
regulated .
These results were confirmed and extended by antiflagellin immunoblot
analysis performed as described previously (16) . The
level of flagellin protein was significantly reduced in the ylxL
mutant LMB231 when compared to levels found in the wild-type strain
(Fig . 3C) . Nonetheless, flagellin protein was easily
detectable in this strain and absent in the sigD null mutant .
Therefore, while the alternate sigma factor encoded by sigD,
D,
is absolutely required for expression of the flagellin gene as
previously determined (11), ylxL appears to
play a critical role in up-regulating its expression .
Motility and chemotaxis function. Having demonstrated that the ylxL mutant is capable of synthesizing flagellin protein, we sought to test whether it is motile and able to demonstrate chemotactic behavior . Semisolid agar (0.3% agar) plates were used as described previously (9) to monitor the ability of the mutant to swim away from the site of inoculation towards areas of greater nutrient concentration . Disruption of the ylxL gene resulted in impaired swimming motility and/or chemotaxis, as demonstrated by a smaller diameter of growth found for this strain when compared to the wild-type strain (Fig . 4A and B) . The ylxL mutant, LMB231, exhibited 40% of the diameter of growth found for the wild-type strain, LMB7, but was clearly capable of swimming motility when compared to two nonmotile strains bearing null mutations in the flagellum-specific alternate sigma factor, sigD, and the flagellar switch protein, fliM .
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The chemotactic efficiency of the ylxL mutant towards three
attractants (asparagine, proline, and mannitol) was monitored by
capillary assays that were performed as previously described (14,
17) . Several concentrations of each attractant were assayed
(data not shown), and the results obtained by use of 10 µM
attractant are presented, since they are consistent with the data
obtained for all concentrations tested (Fig . 4C) . Accumulation
of ylxL mutant cells in capillaries containing the three
attractants tested ranged from 37 to 52% of the number of wild-type
cells that accumulated under identical conditions . While this could
be interpreted as compromised chemotactic behavior, a similar
difference in the level of accumulation (47%) was found for the blank
that contained no attractant (Fig . 4C) . Therefore,
our results suggest that the reduced diameter of growth found on the
semisolid agar plates (Fig . 4A and B) was due to compromised
swimming motility and not a defect in chemotaxis . This result
is consistent with a role of the ylxL gene product in enhancing
D
function . The genes for flagellin and the motility proteins (11,
12) are
D
dependent, while most of the chemotaxis genes are found in the
fla/che operon (1) and are not dependent on the
alternate sigma factor for expression (16) .
Autolysin activity. The genes for several autolysins contain
D-dependent
promoters (4, 8), and cells
lacking
D
function grow as long filaments due to decreased autolytic activity
necessary for cell separation (9) . Microscopic
observation of the ylxL mutant compared to the wild-type
strain demonstrated filamentous growth, but the filaments were
significantly shorter than those found for the sigD null
mutant and included a mixture of short and long filaments (Fig.
5) . These data further support the postulate that the
ylxL gene product is required for maximal
D
activity in the cell .
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Summary. In this work we have demonstrated that the ylxL
gene is the last gene of the fla/che operon that apparently
encodes a protein required for maximal
D
function . Sequence analysis of the intergenic region between ylxL
and rpsB has failed to detect a factor-independent
transcriptional terminator, suggesting that the termination of
transcription identified in this study is factor dependent . The
location of ylxL at the end of the fla/che transcription
unit, coupled with its demonstrated role in enhancing
D
function and read-through into rpsB, provides for the
intriguing possibility that it acts as a sensor for complete
fla/che transcription and/or couples flagellin transcription and
translation .
While BLAST analysis using the predicted protein product of
ylxL as the query sequence showed only weak matches to proteins
of unknown function, a search of the B . subtilis genome using
products of several enteric flhC genes as the query identified
a single match, the ylxL gene product (2) . The flhC
gene is part of the master operon (7) that is
required for expression of the flagellar regulon in enteric bacteria .
This gene encodes part of a transcriptional activator complex
absolutely required for expression of the genes that form the HBB and
the flagellum-specific sigma factor in that system (6) .
However, ylxL does not appear to be required for sigD
expression, since a strain disrupted for ylxL in this study
was able to display limited
D
functions . Interestingly, the similarity between the ylxL gene
product and the enteric FlhC proteins is limited to a third of the
protein and suggests a different domain structure . Nonetheless, the
ylxL gene product may function as a transcriptional activator
of
D-dependent
genes . Further, its unique location at the end of the fla/che
transcription unit may allow this activity to be coupled to complete
transcription of the operon . Finally, read-through from ylxL
to rpsB may suggest a link between
D-dependent
transcription of the hag gene and its subsequent translation .
Recent work from the Hughes laboratory (P . Aldridge, H . R . Bonifield, J . Gnerer, J . E . Karlinsey, and K . T . Hughes, Abstr . BLAST VII Bact . Locomotion Signal Transduction, p . 42, 2003) suggests that hag mRNA in Salmonella enterica serovar Typhimurium may be targeted to the HBB complex for translation . The product of the rpsB gene encodes a component of the ribosome found to be localized to the poles of cells (5) . The genetic organization of this gene immediately downstream of ylxL and the low level of read-through found in this study suggest such a link, but much greater investigation is required to support this prediction .
| ACKNOWLEDGMENTS |
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This research was supported by NSF-RUI grant MCB-0112949 to L.M.-M.,
DOE-GAANN P200A80128 support to H.W., NIH-MBRS RISE grant GM5-59298
to P.L., and RO1 GM54365 to G.W.O .
We are grateful to Peter Ingmire for critical reading and editing of the manuscript .
| FOOTNOTES |
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* Corresponding author . Mailing address: Department of Biology,
San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132 .
Phone: (415) 338-1548 . Fax: (415) 338-0927 . E-mail: marquez@sfsu.edu .
| REFERENCES |
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D-dependent
functions in Bacillus subtilis. J . Bacteriol . 172:3435-3443.
28
form of RNA polymerase . J . Bacteriol . 174:3095-3101.
D
form of RNA polymerase . J . Bacteriol . 174:4197-4204.
D-dependent
gene expression . J . Bacteriol . 182:3055-3062 .
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