62NISR Research GRANT
2008 Young Investigator Research Grant

Regulation of genes expression on Escherichia coli genome
with communications among microbes
Kaneyoshi YAMAMOTO
Department of Frontier Bioscience, Hosei University

Research aims
Bacteria mostly live in communities composed of manyother microorganisms and often in association with higherorganism hosts, such as animals, plants, and insects. Individual bacteria must alter their behaviour through interaction between microorganisms or interaction between bacteria and host to maintain their survival in complex naturalenvironments. The chemical interaction between bacterial
cells is known to be quorum sensing (QS), by which bacterial population-dependent response is controlled through diffusible molecules produced by individual cells. The QS has
been found to be involved not only in interactions between
cells of the same bacterial species, but also involved in interactions between cells of different bacterial species or between bacterial cells and their host cells. Here we attempted
to identify Escherichia coli genomic functions related tocommunications between E. coli cells or communications
between cells of E. coli and another bacterial species, Bifidobacterium longum.
N-acetylymuramic acid (MurNAc), a component of E.
coli cell wall, can be used as a carbon source by E. colicells.1) The murQP operon of E. coli encodes the enzymes
for the degradation of MurNAc. The operon is located next
to the yfeT gene on the E. coli genome, each being transcribed into opposite directions. In this study, YfeT wasidentified as a regulator for the murQP operon.1,2) Furthermore, we identified E. coli promoters induced only when E.
coli was cocultured with B. longum.
Methods
For measurement of the activity of yfeT and murQ promoters, we constructed yfeT-lacZ and murQ-lacZ protein
fusions. The lacZ fusion genes on the plasmid were inserted
into l RS45 phage by recombination. Using the recombinant lambda phages, lysogens of E. coli BW25113 (parentstrain) and DI0200 (D yfeT ) were constructed: [DI0101(BW25113, l murQ-lacZ); DI0102 (BW25113, l yfeTlacZ ); DI0201 (DI0200, l murQ-lacZ); and DI0202(DI0200, l yfeT-lacZ)]. The lysogens were grown in M9glycerol medium and then subjected to measurement of b galactosidase activity.2)
Construction of the promoter collection of E. coli K-12
W3110 strain is being carried out in PPT (Promoter ProjectTeam) using pGRP as a vector. The PPT collection set con-tains about 1000 plasmid clones in which each E. coli promoter was translationally fused with the gfp (green fluorescent protein) reporter gene. Each E. coli transformant harboring one of the PPT plasmid clones was grown with andwithout B. longum in ABCM medium in a well on a 96well plate under anaerobic condition and then the intensityof GFP in each well was measured.
Results
Transcriptional regulation of enzymes for N-acetylymuramic acid degradation in E. coli.
The lysogens, DI0101, DI102, DI0201, and DI0202,
were grown in M9-glycerol medium at 37°C. At stationary
phase (OD 600 nm1.4), cultures were subjected to measurement of b -galactosidase activity. The high-level expression of murQ-lacZ and yfeT-lacZ was detected only in theyfeT deficient mutant (Fig. 1A). Jaeger and Mayer recently
reported that YfeT is indeed involved in repression of themurQ promoter and proposed to rename it to MurR.3) In
good agreement with their report, our finding confirmedthat YfeT represses transcription of both murQP and yfeToperons. MurNAc is transported and converted to MurNAc6-phosphate by MurNAc-specific membrane component(MurP) of MurNAc-specific enzyme IIBC PTS (phosphotransferase system). The addition of MurNAc and MurN induced the expression of both murQ-lacZ and yfeT-lacZ inwild-type E. coli (Fig. 1B), supporting the prediction thatMurNAc-6-phosphate is the inducer of YfeT in vitro.3)
Identification of E. coli promoters induced in co-culture
with B. longum.
Each of the approximate 1,000 PPT plasmid clones, eachof which contains a different E. coli promoter fused to thegfp reporter gene, was transformed into E. coli DH5a . E.
coli transformants obtained and B. longum were separately
grown in ABCM medium at 37°C under anaerobic condition. Then, each of E. coli culture (5104 cells) was mixedwith B. longum culture (4.95106 cells) in fresh ABCM
medium and then incubated at 37°C for 20 hours underanaerobic condition. The fluorescence intensity of each culture was measured with FL600 (Bio Tek) and plotted in thescatter plot (Fig. 2). Twenty-eight E. coli promoters wereinduced in co-culture with B. longum. Finally four promoters, lysC, macA, proX, and ygaU, were confirmed to be induced in a manner dependent on number of B. longum cells

63Fig. 1. Repression of murQ and yfeT expressions by YfeT in an MurNAc-dependent manner. [A] murQ (left) and yfeT (right) expression in E. coli wildtype (black bar) and D yfeT (white bar). [B] The effect on murQ (left) and yfeT (right) expressions generated by addition of MurNAc, MurN, GalNAc, andGalN in E. coli.

ured. Among four promoters, only ygaU promoter was induced by CMs of B. longum. These results suggested thatygaU is induced by some factor(s) in B. longum CM andthe remaining three promoters, lysC, macA, and proX, seemto be induced by an as-yet-unidentified mechanism operating in cell-cell communication between cells of E. coli andB. longum.
Conclusion

Fig. 2. The expression profile of E. coli promoters with and without B.
longum. The intensity of fluorescence from promoters was measured inculture with (vertical line) and without (horizontal line) B. longum andspotted. Red circle indicates the induced promoters in co-culture with B.
longum.

present in the stationary cultures.
E. coli promoters induced only in the presence of B. longum
cells.
One possible mechanism of promoter induction in coculture with B. longum is that small molecule secreted by B.
longum is a signal triggering gene expression from the promoters, as is the case in QS system. If this is the case, conditioned medium (CM) of B. longum must induce the identified E. coli promoters even in the absence of B. longum
living cells. B. longum was grown to stationary phase andpelleted by centrifugation. The supernatant was recovered
and pH was adjusted to 7.2 with NaOH. The solution wassupplemented with LB medium (final conc., 0.25LB),
and then sterilized by filtration through a 0.22 m m filter togenerate CM. Each E. coli transformant carrying the lysC,macA, proX, or yagU promoter fused to the gfp gene wasgrown in CM and the intensity of fluorescence was meas-Molecular mechanism for communication between E. colicells
MurNAc is a component of E. coli cell wall. The E. coli
murQP operon encoding enzymes for MurNAc degradation
is induced by MurNAc or MurN, which is derived fromdead E. coli cells.4) Thus, the regulatory system for expression of E. coli murQP operon could represent a mechanism
for communication between cells of E. coli, by which E.
coli living cells can effectively utilize MurNAc molecules
derived from dead cells of E. coli in natural environments.
Molecular mechanism for communication between E. coliand B. longum cells
Three E. coli promoters, lysC, macA, and proX, were induced only in the presence of living cells of B. longum,
suggesting that a novel molecular mechanism might be operating in communication between E. coli and B. longumcells.
References
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Jaeger, T., M. Arsic, and C. Mayer. 2005. Scission of the lactyl etherbond of N-acetylmuramic acid by Escherichia coli “etherase.” J.
Biol. Chem. 280: 30100–30106.
Ishii, D., Ishihama, A., and Yamamoto, K. 2009. Two Modes of Autoregulation of the murR Repressor in Escherichia coli. Biosci.
Biotechnol. Biochem. 73: 2528–2530.
Jaeger, T., and C. Mayer. 2008. The transcriptional factors MurR andcatabolite activator protein regulate N-acetylmuramic acid catabolism in Escherichia coli. J. Bacteriol. 190: 6598–6608.
Goodell, E. W. 1985. Recycling of murein by Escherichia coli. J.
Bacteriol. 163: 305–310.