YmfM, a putative d homologue in Bacillus subtilis

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YmfM, a putative d homologue in Bacillus subtilis
Lee Katz and Dr. Charles P. Moran Jr. Department
of Microbiology and Immunology Emory University
School of Medicine Atlanta, GA 30322
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Introduction

• In Bacillus subtilis, d is a subunit of RNA
  polymerase (RNAP) and is encoded by the rpoE
  gene. One suggested role of d is to provide
  promoter selectivity to the core RNAP.
• Given that d is a subunit of RNAP, it was
  initially thought to be an essential gene.
  However, a d mutant is viable and shows the same
  growth pattern as that of the wild-type (López de
  Saro et al. 1995).
• One possible explanation could be the presence of
  a d homologue in the genome.
• To check for a homologue, we searched the
  Bacillus genome for closely related genes to d.
  The closest match resulted in the identification
  of a protein YmfM, which did not have a known
  function.
• In an amino acid sequence alignment, YmfM shows a
  20 amino acid sequence identity to d (Fig. 1).
• We hypothesized that if YmfM was indeed a true
  homologue of d, then inactivation of ymfM would
  affect gene transcription in the same way that
  the inactivation of d would.

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• In order to test the hypothesis that ymfM is a
  homologue of d, I decided to knock out the ymfM
  gene.
• The technique that I will use to knock out ymfM
  is called homologous recombination (Fig. 2).

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RpoE MSLDDLQAATKIQKRYLTALEEGNYDIIPGKVLCSHSSSNMQKPL
ANDADQLFEEHKKDI 60 .. . . .
. .. . . YmfM
MGI----------KQY--SQEELK-----------------EMALVEIAH
ELFEEHKKPV 31 RpoE PNTYHDDVSEKISGMNLQKEMPKPASKAL
ELLPTILVILGVIVVIA-IVYAIIQFGNHKN 119 ...
.. .... . . . ..... .... ..
. . YmfM P--FQELLNEIASLLGVKKE---ELGDRIAQFYTDLNIDGR
FLALSDQTWGLRSWYPYDQ 86 RpoE SDDHNAGSEKAITQSESKYE
IPKDSTLKENQNNSSEKETDTKKETKENEDKKKEN-DSEK 178
... . .... . . .. . . ...
. .. YmfM LDEETQPTVKA-KKKKAKKAVEEDLDLDEFEEI
D-EDDLDLD-EVEEELDLEADDFDEED 143 RpoE
LEINAAGTEGSLTTYEVSGADKIDLELKASDSSWI 213 . .
. . . . YmfM
LDEDDDDLE-----IEEDIIDEDDEDYDDEEEEIK 173
Figure 1 Amino acid sequence alignment of RpoE
(d) and YmfM. The alignment was generated by
using the software ALIGN (Genestream). Two dots
represent identical amino acid, and one dot
represents similar amino acids.
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Creation of gene-specific deletions in Bacillus
subtilis
In homologous recombination, a plasmid is
constructed such that it has two regions of
homology surrounding a selectable marker. The
regions of homology are sequences of DNA that are
practically identical to each other. For
example in Figure 2, region A on the plasmid is
almost identical to region A on the chromosome.
When a double crossover occurs, the gene of
interest is switched with the marker from the
plasmid.
Figure 2 Products of homologous recombination, a
powerful tool to create genespecific
recombination
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Step 1 PCR the flanking regions of ymfM
A)
ymfL
ymfM
pgsA
NcoI
XhoI
XbaI
EcoRI
Figure 3 Location of the ymfM locus on the
chromosome of Bacillus subtilis and the genes
flanking it. A portion of the figure was
generated by the SubtiList database. The two
flanking genes are the regions of homology (Fig.
2). A) The arrows indicate relative positions
of primers used in polymerase chain reaction
(PCR) to amplify chromosomal DNA. The ymfL
primers had XhoI and EcoRI recognition sequences,
and the pgsA primers had NcoI and XbaI
recognition sequences inserted at their 5and
3-ends, respectively. B) DNA agarose gel
showing the amplification products of the
flanking loci. Lane a and b were the PCR products
of the ymfL gene Lanes d and e, PCR product of
pgsA. Lanes c and f had no DNA, as a control.
B)
a b c d e f
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Step 2 Insert the PCR products into pLitmus 28.
The two flanking loci were inserted into the
plasmid pLitmus 28. E. coli were transformed
with the new plasmid. Then the cloned plasmids
were purified and screened by cleavage enzymes
XhoI and EcoRI (Fig. 4A) and by cleavage enzymes
NcoI and XbaI (Fig. 4B). The cleavage products
were photographed after electrophoresis in 1
agarose gel (in panels A and B). Each gel has a
molecular weight marker that helps to
approximate the size of the bands of DNA. The
comparison between the bands and the marker helps
determine whether or not the plasmid contains
the insert, since the size of the actual insert
was already known.
A)
Figure 4 Screening for inserts (A) Lanes 1 and
2, both took up the plasmid with the ymfL gene,
which is 1000 base pairs (bp). Lane 3, pLitmus
28, and lane 4, the PCR product ymfL. (B) Lanes
3, 4, 12, 18, and 20 all took up the pgsA PCR
product, which is 900 bp. The control was the
plasmid with the first insert. (C) What a marker
looks like. Each band shows at which position
any sized DNA fragment would be.
C)
B)
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Step 3 Inserting a selectable marker
BglII
BamHI
cmr
XbaI
XhoI
BamHI
NcoI
EcoRI
Figure 5 Inserting the marker (Chloramphenicol
resistance gene). Step 3 A selectable
marker will be added between the other two
inserts, in the plasmid. It will be a cmr gene,
which codes for chloramphenicol resistance. I
have not finished this step yet. In the near
future, this clone will be made, and the mutant
will be isolated.
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References

• Genestream. http//xylian.igh.cnrs.fr/bin/align-g
  uess.cgi
• López de Saro, Francisco J. Woody, A-Young Moon
  and Helmann, John D. Structural Analysis of the
  Bacillus subtilis d Factor A Protein Polyanion
  which Displaces RNA from RNA Polymerase. J. Mol.
  Biol. 1995, pp. 189-202.
• SubtiList Web Server. http//genolist.pasteur.fr/
  SubtiList

Acknowledgements
I thank Dr. Amrita Kumar, who taught me most of
the laboratory techniques that I know and who set
me in the right direction in this experiment.
Also appreciated are the others who were around
when I needed to ask a question Bill Blaylock,
Denine Crater, Chadia Toukoki, and Jason Opdyke.
This work was supported by the National
Institutes of Health (NIAID) training grant
"Molecular Mechanisms of Microbial Pathogenesis,"
5T32 AI07470