Transport and Catabolism of Pentitols by Listeria monocytogenes

12 Pages·2016·0.29 MB·English

Research Article
J Mol Microbiol Biotechnol 2016;26:369–380 Received: May 5, 2016
DOI: 10.1159/000447774 Accepted: June 22, 2016
Published online: August 24, 2016
Transport and Catabolism of Pentitols
by Listeria monocytogenes
Takfarinas Kentache a Eliane Milohanic a Thanh Nguyen Cao a

Abdelhamid Mokhtari a, b Francine Moussan Aké a Que Mai Ma Pham a

Philippe Joyet a Josef Deutscher a, c

a Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas , France; b Department of Biology,

8 May 1945 University, Guelma , Algeria; c Centre National de la Recherche Scientifique, UMR8261 (affiliated with

Univ. Paris Diderot, Sorbonne Paris Cité), Expression Génétique Microbienne, Institut de Biologie Physico-Chimique,
Paris , France

Key Words the EIIC-encoding gene ( lmo0508) affected neither D -arabi-
Listeria monocytogenes · D -Arabitol utilization · D -Xylitol tol nor D -xylitol utilization, although D -arabitol induces the
utilization · Phosphotransferase system · Virulence gene expression of this operon. Both operons are controlled by
repression MtlR/LicR-type transcription activators (Lmo2668 and
Lmo0501, respectively). Phosphorylation of Lmo0501 by the
soluble PTSA xl components probably explains why D -arabitol
Abstract also induces the second pentitol operon. Listerial virulence
Transposon insertion into L isteria monocytogenes lmo2665 , genes are submitted to strong repression by PTS sugars,
which encodes an EIIC of the phosphoenolpyruvate:carbo- such as glucose. However, D -arabitol inhibited virulence
hydrate phosphotransferase system (PTS), was found to pre- gene expression only at high concentrations, probably ow-
vent D -arabitol utilization. We confirm this result with a dele- ing to its less efficient utilization compared to glucose.
tion mutant and show that Lmo2665 is also required for D - © 2016 S. Karger AG, Basel
xylitol utilization. We therefore called this protein EIICA xl .
Both pentitols are probably catabolized via the pentose
phosphate pathway (PPP) because l mo2665 belongs to an Introduction
operon, which encodes the three PTSA xl components, two
sugar-P dehydrogenases, and most PPP enzymes. The two D -Ribitol, D -xylitol, and D -arabitol are the three major
dehydrogenases oxidize the pentitol-phosphates produced naturally occurring pentitols. D -Arabitol (also called D -
during PTS-catalyzed transport to the PPP intermediate xy- arabinitol and D -lyxitol) is relatively abundant in nature
lulose-5-P. L . monocytogenes contains another PTS, which and is produced by lichens and fungi. Bacteria from most
exhibits significant sequence identity to PTSA xl . Its genes are phyla, such as the proteobacteria K lebsiella pneumoniae
also part of an operon encoding PPP enzymes. Deletion of [Heuel et al., 1997] and P seudomonas fluorescens [Brün ker
© 2016 S. Karger AG, Basel Eliane Milohanic or Josef Deutscher
1464–1801/16/0266–0369$39.50/0 Micalis Institute
INRA, AgroParisTech, UMR1319
E-Mail [email protected]
FR–78850 Thiverval-Grignon (France)
www.karger.com/mmb
E-Mail eliane.milohanic @ grignon.inra.fr or Josef.Deutscher @ grignon.inra.fr
et al., 1998], the actinobacterium C orynebacterium glu- able to utilize D -ribitol and D -xylitol. They contain D -ri-
tamicum [Laslo et al., 2012], and the firmicutes E nterococ- bitol-5-P and D -xylitol-5-P dehydrogenases [Hausman
cus avium [Povelainen et al., 2003] and L isteria monocyto- and London, 1987], which suggested that these pentitols
genes [Saklani-Jusforgues et al., 2001] utilize D -arabitol as are taken up by PTSs. Indeed, an EII component specific
a carbon and energy source. The γ-proteobacterium K . for xylitol was purified from L. casei strain Cl83 [London
pneumoniae and the actinobacterium C . glutamicum take and Hausman, 1983]. In addition, the D -ribitol-specific
up D -arabitol via closely related transporters of the major mannose-type PTS of L. casei , which contains two mem-
facilitator superfamily (DalT and RbtT, respectively). D - brane-spanning components (EIIC Rtl and EIIDR tl ), has
Arabitol 4-dehydrogenases with significant similarity to recently been characterized [Bourand et al., 2013]. Inter-
mannitol-1-P dehydrogenases subsequently oxidize intra- estingly, D -xylitol-5-P dehydrogenase purified from L .
cellular D -arabitol to D -xylulose [Heuel et al., 1997]. The casei was also able to efficiently oxidize D -arabitol-5-P
γ-proteobacterium P . fluorescens transports D -arabitol via [Hausman and London, 1987], which is one of the two
an ABC transporter of broad substrate specificity, which possible products of PTS-catalyzed transport and phos-
also recognizes D -mannitol and D -glucitol. The ABC phorylation. However, no L . casei strain has been report-
transporter is composed of the binding protein MtlE, the ed to utilize D -arabitol. In fact, L. monocytogenes was the
two membrane-spanning transport proteins MtlF and first firmicute for which PTS-catalyzed transport of D -
MtlG, and the ATP hydrolyzing protein MtlK [Brünker et arabitol was suggested [Saklani-Jusforgues et al., 2001].
al., 1998]. A gene encoding a polyol dehydrogenase (MtlD) Later, E. avium was reported to possess a D -arabitol-
is also present in the P . fluorescens operon. MtlD has the phosphate dehydrogenase, ApdH, which converts both
highest affinity for D -arabitol and converts the pentitol to possible products of PTS-catalyzed D -arabitol phosphor-
D -xylulose. With lower efficiency it transforms D -manni- ylation, D -arabitol-1-P and D -arabitol-5-P, into the pen-
tol and D -glucitol into D-fructose [Brünker et al., 1997]. tose phosphate pathway (PPP) intermediates D -xylulose-
In contrast to proteobacteria and actinobacteria, fir- 5-P and D -ribulose-5-P, respectively [Povelainen et al.,
micutes transport pentitols via a phosphoenolpyruvate: 2003]. The apdH gene is part of an operon including also
glycose phosphotransferase system (PTS) either of the ga- the genes for a PTS of the galactitol family ( fig. 1 c). ApdH
lactitol or the mannose family. The galactitol PTSs are usu- exhibits significant similarity to the L. monocytogenes
ally composed of two cytoplasmic substrate-specific pro- proteins Lmo2663 and Lmo2664, the genes of which are
teins (EIIA and EIIB) and the integral membrane protein also part of an operon encoding a galactitol type PTS
EIIC. PTSs of the mannose family contain a second mem- ( fig. 1a ). Interestingly, insertion of a transposon into
brane protein, EIID. Together with the general PTS com- lmo2665 , which encodes an EIIC of the galactitol family
ponents EI and HPr, the two soluble carbohydrate-specif- [Barabote and Saier, 2005], prevented L. monocytogenes
ic proteins form a phosphorylation cascade. In the first from fermenting D -arabitol [Saklani-Jusforgues et al.,
step, EI autophosphorylates at a conserved histidyl residue 2001].
by using phosphoenolpyruvate as phosphoryl donor [Al- L. monocytogenes possesses a second operon encoding
pert et al., 1985]. P∼ EI transfers the phosphoryl group to an ApdH-like protein (Lmo0505) and a potential pentitol
His-15 in HPr, and P∼ His-HPr phosphorylates one of PTS. We therefore decided to characterize pentitol trans-
usually several different carbohydrate-specific EIIA pro- port and metabolism in this pathogen in more detail. We
teins present in a bacterium. P ∼ EIIAs, which are also also studied the effect of D -arabitol utilization on viru-
phosphorylated at histidine residues [Deutscher et al., lence gene expression. Glucose, which is mainly metabo-
1982], usually transfer the phosphoryl group to a con- lized via the PPP [Eisenreich et al., 2006], and other PTS
served Cys residue of their cognate EIIB [Volpon et al., substrates strongly repress PrfA-regulated virulence gene
2006]. Only EIIBs of the mannose family are phosphory- expression [Milenbachs et al., 1997; Park and Kroll, 1993;
lated at a histidyl residue. In the last step, P∼ EIIB donates Scortti et al., 2007]. The non-PTS carbon sources glycerol
its phosphoryl group to the carbohydrate bound to the cy- [Zébré et al., 2015] and maltose [Deutscher et al., 2014a]
toplasmic side of the corresponding membrane-spanning have no repressive effect. PTS components were therefore
EIIC [Cao et al., 2011]. Phosphorylation of the carbohy- thought to play a role in carbohydrate-mediated PrfA
drate probably lowers its affinity for the EIIC and the phos- regulation [Herro et al., 2005; Mertins et al., 2007]. Be-
phorylated carbohydrate is released into the cytoplasm. cause D -arabitol is a PTS substrate probably also mainly
The first evidence for PTS-catalyzed transport of pen- metabolized via the PPP, we tested whether D -arabitol
titols came from studies with L actobacillus casei strains would also exert virulence gene repression.
370 J Mol Microbiol Biotechnol 2016;26:369–380 Kentache/Milohanic/Cao/Mokhtari/Aké/
DOI: 10.1159/000447774 Ma Pham/Joyet/Deutscher
2660 2665
3-EPI TK A-ISO R-ISO 2663 2664 EIICAxl EIIAAxl TR
cre site
a 2659 (cid:169) 2661 2662 DH DH (cid:169) EIIBAxl 2667 2668
R-ISO (cid:169) 0501 0503 0508
0498 A-ISO TR ? EIIA ? 3-EPI 0506 EIIB EIIC
cre site 0499 0500 Ins 0502 Ins 0504 0505 DH 0507 (cid:169)
b TA
I570_00051
TR EIIA EIIB EIIC ApdH TR EIIA EIIB EIIC
c I570_00048 I570_00052 I570_00053 I570_00056 DH
Fig. 1. Gene organization of the two D -arabitol-induced operons lmo0502 and 0504 encode proteins of unknown function. Δ indi-
in L. monocytogenes (a , b ) and the D -arabitol operon of E . avium cates the genes which were deleted and ‘Ins’ those which were dis-
(c ). The genes encoding the PTS proteins are presented as grey ar- rupted by plasmid insertion. Bars represent the regions amplified
rows and the pentitol dehydrogenases as black arrows. A-ISO = during qRT-PCR experiments. The numbers in a and b indicate
Arabinose-5-P isomerase; DH = D -arabitol-1-P/D-xylitol-5-P de- the gene names ( lmo2659 , etc.) of strain EGD-e on the ListiList web
hydrogenases; 3-EPI = ribulose-5-P 3-epimerases; R-ISO = ribu- server. The numbers in c correspond to the locus tag (I570_00048,
lose-5-P isomerases; TK = transketolase; TA = transaldolase; TR = etc.) of E . avium strain ATCC 14025.
PRD-containing MtlR-like transcription activators. The genes
Results and Discussion Two Pentitol-P Dehydrogenases Are Encoded by the
Presumed D -Arabitol Operon lmo2668–lmo2659
Deletion of the EIIC-Encoding lmo2665 Gene Prevents To determine whether the genes downstream from
D -Arabitol Utilization lmo2665 ( fig. 1 a) encode enzymes involved in D -arabitol
The previously reported insertion of a transposon into metabolism, we purified several of them as described in
lmo2665 ( fig. 1 a), which encodes an EIIC PTS component Experimental Procedures (online suppl. fig. S1; see www.
of the galactitol family, caused a total loss of D -arabitol karger.com/doi/10.1159/000447774 for all online suppl.
fermentation [Saklani-Jusforgues et al., 2001]. In order to material). We first tested the activity of the two enzymes
avoid the polar effects associated with the transposon in- Lmo2663 and Lmo2664, which exhibit significant iden-
sertion, we constructed a mutant carrying an in-frame tity (up to 65%) to the E . avium D -arabitol-phosphate de-
deletion of the l mo2665 gene. Identical to the transposon hydrogenase ApdH, which reduces D -xylulose-5-P to D -
insertion mutant, the Δl mo2665 mutant had also lost the arabitol-1-P and D -ribulose-5-P to D -arabitol-5-P [Pove-
capacity to grow on D -arabitol as the sole carbon source lainen et al., 2003]. The two listerial enzymes reduce
( fig. 2 c), but grew normally on glucose and glycerol D -xylulose-5-P ( table 1 ), but only Lmo2664 was also able
( fig. 2 a, b). This result suggested that Lmo2665 is the main to slowly reduce D -ribulose-5-P ( fig. 3 ; table 1 ). Reduc-
protein catalyzing the uptake of D -arabitol. A Δp tsI mu- tion of D -ribulose-5-P by Lmo2664 must lead to the for-
tant (lacks the general PTS component EI) [Aké et al., mation of D -arabitol-5-P because the enzyme was not
2011] was also not able to grow on D -arabitol (data not able to oxidize D -ribitol-5-P to D -ribulose-5-P. The activ-
shown), thus confirming the PTS-mediated D -arabitol ity of Lmo2664 therefore strongly resembles that of ApdH
uptake. [Povelainen et al., 2003]. However, in contrast to ApdH,
none of the two listerial enzymes could use NADPH as a
reducing substrate. The addition of 10 m M EDTA to the
assay mixture almost completely inhibited the dehydro-
Pentitol Utilization by L isteria J Mol Microbiol Biotechnol 2016;26:369–380 371
monocytogenes DOI: 10.1159/000447774
1.3
1.4
1.0
25 mM 25 mM 1.1 25 mM D-arabitol
glucose 1.2 glycerol
0.8 0.9
1.0
D6000.6 D6000.8 D600 0.7
O O O 0.5
0.6
0.4
EGD-e
0.4 EGD-e 0.3
EGD-e lmo0508
0.2 lmo0508 0.2 lmo0508 0.1 lmo2665
lmo2665
lmo2665
0 0 –0.1
0 20 0 10 20 30 0 10 20 30 40
a Time (h) b Time (h) c Time (h)
Fig. 2. Growth of L . monocytogenes strains on various carbon either glucose ( a ), glycerol (b ), or D -arabitol (c ). While the three
sources measured with a BioTek microplate reader. The L. mono- strains grew nearly identical in glucose- and glycerol-containing
cytogenes wild-type strain EGD-e and the Δl mo0508 and Δ lmo2665 media, the Δ lmo2665 mutant had lost the capacity to grow on D -
mutants derived from it were grown in MM containing 25 m M of arabitol.
Table 1. Enzyme activities of purified proteins
Gene Enzyme activity measured Specific activity,
μmol/min and mg protein
lmo0499 D-Arabinose-5-P isomerase 0.23
lmo0505 D-Ribulose-5-P 3-epimerase 1.27
lmo0506 D-Xylulose-5-P 2-reductase (Zn2+)a 0.19
lmo0506 D-Ribulose-5-P 2-reductase (forms D-ribitol-5-P) 0.07
lmo2659 D-Ribulose-5-P 3-epimerase 4.91
lmo2661 D-Arabinose-5-P isomerase 0.07
lmo2662 D-Ribose-5-P isomerase 4.21
lmo2663 D-Xylulose-5-P reductase (Mn2+) (forms D-arabitol-1-P and/or D-xylitol-5-P) 3.87
lmo2664 D-Xylulose-5-P reductase (Mn2+) (forms D-arabitol-1-P and/or D-xylitol-5-P) 6.32
lmo2664 D-Ribulose-5-P 2-reductase (Mn2+) (forms D-arabitol-5-P) 0.08
a Metal ions are mentioned when Mg2+ was replaced in the assay mixture with either Mn2+ or Zn2+.
genase activities of the two enzymes, thus confirming the PPP. They are usually interconverted in response to the
dependence of the two pentitol-P dehydrogenases on di- metabolic need of the cell. Similar to the E. avium EIIC,
valent cations. Similarly as observed for E. avium ApdH, Lmo2665 probably phosphorylates D -arabitol mainly to
Mn 2+ was found to be the preferred cofactor because in D -arabitol-1-P, which is oxidized to D -xylulose-5-P. Be-
the presence of Mg 2+ the initial activity of the two liste- cause the metabolism via the PPP requires one D -ribu-
rial enzymes was about 10-fold lower (data not shown). lose-5-P and two D -xylulose-5-P molecules in order to
produce one glyceraldehyde-3-P and two fructose-6-P
Several PPP Enzymes Are Encoded by the D -Arabitol molecules, a significant part of D -xylulose-5-P formed
Operon during growth on D -arabitol needs to be converted to D -
The two P-ketopentoses formed by the dehydrogenase ribulose-5-P. Lmo2659 exhibits similarity to D -ribulose-
Lmo2664, D -ribulose-5-P and D -xylulose-5-P, fuel the 5-P 3-epimerases, which catalyze the interconversion of
372 J Mol Microbiol Biotechnol 2016;26:369–380 Kentache/Milohanic/Cao/Mokhtari/Aké/
DOI: 10.1159/000447774 Ma Pham/Joyet/Deutscher
The gene l mo2660 in the presumed D -arabitol operon
encodes probably a transketolase (f ig. 1 ), another key en-
Addition of Lmo2664
zyme of the PPP, which converts D -ribose-5-P and xylu-
0
D-ribulose-5-P lose-5-P into glyceraldehyde-3-P and sedoheptulose-7-P.
However, deletion of this protein did not prevent growth
–0.5
on D -arabitol as the sole carbon source (data not shown).
A likely explanation for this result is the presence of at
–1.0
least two other predicted transketolase-encoding genes in
0
D34–1.5 the L. monocytogenes genome ( lmo1305 and lmo0342 ),
O which can probably compensate for the deletion of
–2.0 lmo2660 .
–2.5 Growth of L. monocytogenes on D -Arabitol Induces
Two Operons
D-xylulose-5-P
The only PPP enzyme lacking in the presumed D -arab-
0 5 10 15 20 25 30 itol operon is transaldolase. However, L. monocytogenes
Time (min)
contains three genes encoding potential transaldolases.
One of them, lmo0500 , is located next to genes encoding
the components of another galactitol-type PTS as well as
Fig. 3. Spectrophotometric assay of the Lmo2664-catalyzed
several enzymes strongly resembling the corresponding
NADH-dependent reduction of D -ribulose-5-P to D -arabitol-5-P
(D -arabitol-5-P 2-dehydrogenase, broken line) and of D -xylulose- proteins of the D -arabitol operon ( fig. 1 b). The strong
5-P to D -arabitol-1-P (D -arabitol-1-P 4-dehydrogenase, solid line). similarity of the protein sequences and the gene organiza-
Assay mixtures were prepared as described in Experimental Pro- tion prompted us to test whether growth on D -arabitol
cedures and preincubated for 12 min at room temperature before
would induce this presumed second pentitol operon. We
the reactions were started by adding Lmo2664 (arrow).
therefore carried out qRT-PCR experiments with primers
based on the genes lmo0500 , lmo0502, or l mo2665, and
RNA prepared from L . monocytogenes EGD-e cells grown
D -xylulose-5-P and D -ribulose-5-P. By carrying out an in minimal medium (MM) containing glycerol, glucose,
appropriate enzyme assay, we could indeed confirm that cellobiose, or D -arabitol. Indeed, all three genes were
Lmo2659 possesses D -ribulose-5-P 3-epimerase activity strongly induced when L . monocytogenes EGD-e grew on
( table 1 ). D -arabitol ( fig. 4 a–c). However, the EIIC PTS component
To enter the PPP, the ketose D -ribulose-5-P needs to Lmo0508 does not seem to contribute to D -arabitol trans-
be transformed to the aldose D -ribose-5-P, a metabolite port because in contrast to Lmo2665 its deletion had no
also used for nucleic acid synthesis. The enzyme ribose- effect on D -arabitol utilization ( fig. 2 c).
5-P isomerase catalyzes the reversible transformation of The genes of both operons were slightly expressed when
D -ribose-5-P to D -ribulose-5-P. By using a spectropho- the cells grew on glycerol ( fig. 4 a–c). However, growth on
tometric assay we could demonstrate that the protein glucose or cellobiose almost completely prevented their ex-
Lmo2662 possesses D -ribose-5-P isomerase activity. pression, indicating that both operons are submitted to
Lmo2661 resembles D -ribulose-5-P 3-epimerases carbon catabolite repression. Indeed, potential catabolite
from other organisms. However, we could not confirm response elements ( cre) , the target site for the catabolite
the predicted function, but instead found that Lmo2661 control protein A (CcpA)/P-Ser-HPr complex [Deutscher
exhibits low D -arabinose-5-P isomerase activity. It there- et al., 2014b], are located at the beginning of the first gene
fore catalyzes a reaction similar to Lmo2662 by convert- of the l mo0498–0508 operon (GTTATTCGTTAGAA)
ing the ketose D -ribulose-5-P into the aldose D -arabi- ( fig. 1 b) and upstream from the first gene of the l mo2668–
nose-5-P. In certain bacteria, such as Agromyces cerinus , 2659 region (TGAAAGCGTGTACA) ( fig. 1 a). In addi-
D-arabinose-5-P serves as precursor for the synthesis of tion, CcpA-independent repression [Deutscher, 2008]
poly(arabitol phosphate)-containing cell wall teichoic might be mediated via the PTS regulation domain (PRD)-
acid [Shashkov et al., 1995]. A physiological role of D - containing MtlR-like transcription activators Lmo0501
arabinose-5-P in listeriae has so far not been demonstrat- and Lmo2668 [Deutscher, 2008], which, as shown below,
ed [Cech et al., 2014]. control the expression of the two operons.
Pentitol Utilization by L isteria J Mol Microbiol Biotechnol 2016;26:369–380 373
monocytogenes DOI: 10.1159/000447774
lmo0500 lmo0502 expression lmo2665 expression
expression
140 100
100
120
80
80
100
el
v
e
n l 60
o 60 80
pti
cri
s
n
Tra 60 40
40
40
20 20
20
0 0 0
a wtl_glmyo0501l_gmloy0503_gly wtl_amtlo0501l_amtlo0503_atl b wt_glc wt_celwt_gly wlt_maotl0501l_mgloy050l1_maotl0503l_gmloy0503_atl c wt_glc wt_celwt_gly lwtm_oat0l501l_mgloy050l1m_oat0l503l_gmloy0503_atl
Fig. 4. Expression of D -arabitol-induced genes in AML73 and var- glycerol; atl = D -arabitol. In all strains, D -arabitol induced the ex-
ious mutants derived from it. qRT-PCR experiments were carried pression of the three tested genes, with the exception of the lmo0501
out with primer pairs specific for lmo0500 (a ), lmo0502 (b ), or mutant, in which the transcription activator of the lmo0498–0508
lmo2665 ( c ). RNA was prepared from the wild-type strain AML73, operon is deleted and therefore only lmo2665 was normally in-
the lmo0501, or the lmo0503 mutant. The strains were grown on duced by D -arabitol.
the indicated carbon source. glc = Glucose; cel = cellobiose; gly =
The D -Arabitol-Specific EIIC Lmo2665 Also
2.0
Transports D -Xylitol
25 mM xylitol
While carrying out fermentation assays with the gal-
1.5 lery API fermentation kit, we noticed that D -xylitol also
EGD-e
(cid:2865)(cid:21)(cid:25)(cid:25)(cid:24) gave a fermentation signal (data not shown). In addition,
0
D601.0 (cid:2865)(cid:19)(cid:24)(cid:19)(cid:27)(cid:3) the wild-type strain EGD-e was able to grow on MM con-
O (cid:2865)(cid:83)(cid:87)(cid:86)(cid:44)
taining 25 mM D -xylitol as the sole carbon source. The
Δ ptsI mutant had lost this capacity ( fig. 5 ), indicating that
0.5
D -xylitol is a PTS substrate. We therefore suspected that
the EIIC encoded by lmo0508 might catalyze the trans-
0
port of D -xylitol. However, the Δl mo0508 mutant grew
0 10 20 30 40 50
Time (h) normally in MM containing D -xylitol. Instead, we ob-
served that the Δ lmo2665 strain had not only lost the ca-
Fig. 5. Growth of L . monocytogenes strains on D -xylitol measured pacity to grow on D -arabitol ( fig. 2 c), but also grew very
with a BioTek microplate reader. The L . monocytogenes wild-type poorly on D -xylitol ( fig. 5 ). These results established that
strain EGD-e and the Δ ptsI , Δ lmo0508 , and Δ lmo2665 mutants
the PTS encoded by the genes lmo2665–2667 transports
derived from it were grown in MM containing 25 m M D-xylitol. both D -arabitol and D -xylitol, and we called it PTS Axl .
While the wild-type strain and the Δ lmo0508 mutant grew on the
pentitol, very slow growth was observed for Δ lmo2665 and no During their uptake via Lmo2665, D -arabitol is probably
growth for the Δp tsI mutant. mainly phosphorylated to D -arabitol-1-P and D -xylitol to
374 J Mol Microbiol Biotechnol 2016;26:369–380 Kentache/Milohanic/Cao/Mokhtari/Aké/
DOI: 10.1159/000447774 Ma Pham/Joyet/Deutscher
D-Arabitol / D-Xylitol
OH OH OH OH
Fig. 6. Proposed model of PTSA xl -catalyzed HO OH HO OH
uptake and phosphorylation of D -arabitol OH OH
and D -xylitol, and their subsequent catabo- Membrane Lmo0508 Lmo2665
EIIC??? EIICAxl
lism via the PPP in L. monocytogenes . The
following enzyme activities were deter-
D-Arabitol-1-P D-Xylitol-5-P
mined: A-Iso, arabinose-5-P isomerase; 2-
Arabitol-1-P
DH, arabitol-5-P 2-dehydrogenase; 4-DH,
Ribitol-5-P Lmo2664, Lmo2663
arabitol-1-P 4-dehydrogenase; 3-Epi, ribu-
4-DH/2-DH
lose-5-P 3-epimerase; R-Iso, ribose-5-P Lmo0506
isomerase. All these enzymes, except arab-
A-Iso
inose-5-P isomerase, serve to catabolize
Lmo0499 Lmo2659
phosphorylated D -arabitol and D -xylitol Arabinose-5-P Ribulose-5-P Xylulose-5-P
Lmo2661?
vwcoihad iectshh seei svP eaPrlaPslo. e Tninzhdyeum c lemesd ow 0bi4ty9h 8 Da–c -0at5irva0ib8tii etrose lig,d ieeonnn--, ADN L m oL2m6o6R02-4Is9 o8? Lm3o-E0p5i05
tical to those of the lmo2669–2658 operon. ARN Ribose-5-P
However, the substrate of the galactitol NAD Pentose phosphate pathway
type PTS composed of Lmo0503, 0507, and
0508 (EIIC) is not known.
D -xylitol-5-P ( fig. 6 ). This dual substrate specificity might cause Lmo0506 was able to catalyze the reverse reaction,
also be the reason why the operon encodes two pentitol- i.e. the oxidation of D -ribitol-5-P. Despite the observed
phosphate dehydrogenases Lmo2663 and Lmo2664. Both low activities, we tested the possibility that Lmo0508
enzymes use NADH + to reduce xylulose-5-P, but the might transport D -ribitol. However, L . monocytogenes
product could be either D -arabitol-1-P or D -xylitol-5-P. was not able to grow on this pentitol as the sole carbon
It is therefore possible that one of the two enzymes pref- source. Therefore, the substrate of the EIIC PTS compo-
erably acts as D -arabitol-1-P dehydrogenase and the oth- nent Lmo0508 remains obscure. It could be one of the less
er as D -xylitol-5-P dehydrogenase. Unfortunately, we abundant L -pentitols, except L -xylitol, which we found
were not able to test this assumption because D -arabitol- cannot be utilized by L. monocytogenes . After its uptake,
1-P and D -xylitol-5-P are not commercially available. the L -pentitol might be converted to the D -isomer by one
of the enzymes of unknown function and subsequently
The lmo0498–0508 Operon Also Encodes a Pentitol-P metabolized via the PPP.
Dehydrogenase and Several PPP Enzymes Interestingly, E . avium also contains two operons en-
In order to get an indication whether the PTS compo- coding very similar galactitol PTSs organized in tandem
nents encoded by the second operon also transport a pen- ( fig. 1 ). Both begin with the gene encoding the PRD-con-
titol, which will subsequently be metabolized via the PPP, taining transcription activator, which is followed by the
we purified Lmo0499, Lmo0505, and Lmo0506. Lmo0499 three genes for the PTS Gat components and in the first
functions as D -arabinose-5-P isomerase and Lmo0505 operon by the gene encoding the previously characterized
as D -ribulose-5-P 3-epimerase ( table 1 ). Lmo0506 exhib- D -arabitol-phosphate dehydrogenase ApdH [Povelainen
its strong sequence similarity to the dehydrogenases et al., 2003]. A similar dehydrogenase is encoded by the
Lmo2663 and Lmo2664. It was indeed able to reduce xy- last gene of the second operon (f ig. 1 ). The substrate of
lulose-5-P, but its activity was significantly lower than the second PTS is not known.
that of Lmo2663 and 2664 ( table 1 ). In addition, it pref-
erentially used Zn 2+ ions instead of Mn 2+ . Similar to D -Arabitol Induction of the lmo0498 to lmo0508
Lmo2664, Lmo0506 also reduced D -ribulose-5-P, but this Genes Requires the EIIA xl Components
reaction was 3-fold slower than reduction of D -xylulose- Both pentitol operons contain a gene coding for an
5-P. In contrast to Lmo2664, it seems to transform D -ri- MtlR/LicR-like PRD-containing transcription activator,
bulose-5-P into D -ribitol-5-P and not D -arabitol-5-P be- Lmo0501 and Lmo2668. The operons regulated by this
Pentitol Utilization by L isteria J Mol Microbiol Biotechnol 2016;26:369–380 375
monocytogenes DOI: 10.1159/000447774
To test the second possibility, we disrupted the EIIA-
actA expression encoding gene l mo0503 ( fig. 1 ). Phosphorylation of PRD-
containing transcription activators by the corresponding
250
EIIA or EIIB component at the negative regulation site
usually leads to their inactivation. Deletion of the EIIA
therefore renders the corresponding PRD-containing
200 transcription activator constitutively active [Bouraoui et
al., 2013; Deutscher et al., 2014b; Joyet et al., 2010]. qRT-
el PCR experiments revealed strong expression of the
v
n le 150 lmo0500 , lmo0502, and lmo2665 genes in the lmo0503
ptio mutant during growth on D -arabitol, but not on glycerol
cri ( fig. 4 a–c). The finding that expression of l mo0500 and
s
an lmo0502 was not constitutive in the mutant lacking the
Tr 100
EIIA and EIIB components Lmo0503 and Lmo0507 sug-
gests that EIIAA xl and EIIB Axl phosphorylate and inacti-
vate not only their proper regulator Lmo2668, but also
50 Lmo0501. A possible explanation for the cross-induction
might be that D -arabitol metabolism requires an enzyme
encoded by the lmo0498 to l mo0508 genes. This could
only be a gene upstream from lmo0501 because disrup-
0
glc cel gly atl tion of this gene had no effect on D -arabitol utilization. A
likely candidate was l mo0500 , which encodes the PPP en-
zyme transaldolase. A transaldolase gene is not present in
Fig. 7. qRT-PCR experiments with the virulence gene a ctA of
the axl operon. However, deletion of l mo0500 did not af-
strain AML73 grown in liquid MM containing 25 m M of the indi-
cated carbon source. The actA gene was poorly expressed in cel- fect growth on D -arabitol (data not shown), probably be-
lobiose- and glucose-grown cells, significantly expressed during cause L. monocytogenes contains three other genes encod-
growth on glycerol and strongly expressed in D -arabitol-metabo- ing potential transaldolases.
lizing cells.
Effect of D -Arabitol Utilization on Virulence Gene
Expression
type of transcription activators are usually induced by the Efficiently catabolized carbohydrates transported by
uptake and phosphorylation of the corresponding PTS the PTS, such as cellobiose, glucose, mannose, and fruc-
substrate, which prevents phosphorylation of the PRD- tose [Stoll and Goebel, 2010], repress the expression of
containing regulator by the cognate EIIA and EIIB com- the L. monocytogenes virulence genes [Milenbachs et al.,
ponents [Deutscher et al., 2014b]. Although induced by 1997; Park and Kroll, 1993]. These carbohydrates are
D -arabitol, the EIIC Lmo0508 does not seem to transport mainly catabolized via the PPP [Eisenreich et al., 2006].
this pentitol. Possible explanations are that Lmo2668 ac- Our results suggest that the PTS substrate D -arabitol is
tivates the expression of both pentitol operons or that also mainly catabolized via the PPP (f ig. 6 ). We therefore
Lmo0501 is regulated by the EIIA Axl /EIIBA xl components. tested whether D -arabitol despite its less efficient utiliza-
To test the first possibility we interrupted the tran- tion compared to glucose ( fig. 2 ) would also inhibit the
scription activator gene l mo0501 ( fig. 1 ), which did not activity of the virulence gene activator PrfA. Strain
affect growth on D -arabitol as the sole carbon source (data AML73 contains the β-D -glucuronidase-encoding g us
not shown). qRT-PCR experiments revealed that while reporter gene fused to the promoter of the PrfA-con-
lmo2665 was strongly induced by D -arabitol ( fig. 4 c), ex- trolled L . monocytogenes virulence gene h ly [Behari and
pression of the lmo0500 gene ( fig. 4 a) and of the inter- Youngman, 1998]. It was grown on solid MM contain-
genic region between lmo0500 and lmo0501 (data not ing 100 μg/ml X-GlcA and various concentrations either
shown) was very low in the lmo0501 mutant. This result of glycerol, glucose, or D -arabitol. AML73 forms white
established that only Lmo0501, but not Lmo2668, is able colonies when grown on glucose (within a concentra-
to activate the expression of the lmo0498 to l mo0508 tion range of 2.5–50 mM ) and blue colonies when grown
genes. on the nonrepressing glycerol (online suppl. fig. S2).
376 J Mol Microbiol Biotechnol 2016;26:369–380 Kentache/Milohanic/Cao/Mokhtari/Aké/
DOI: 10.1159/000447774 Ma Pham/Joyet/Deutscher
Table 2. Bacterial strains and plasmids used in this study
Genotype and/or property(ies) Reference or source
Strains
L. monocytogenes
AML73 Derivative of strain 10403S; Φ(Phly-gus), serotype 1/2a [Milenbachs Lukowiak et al., 2004]
EGD-e L. monocytogenes reference strain, serotype 1/2a [Glaser et al., 2001]
EGD-e Δlmo0500 This work
EGD-e lmo0501 This work
EGD-e lmo0503 This work
EGD-e Δlmo0508 This work
EGD-e Δlmo2660 This work
EGD-e Δlmo2665 This work
EM1001 AML73 ΔptsI (lmo1003) [Aké et al., 2011]
FA1019 AML73 ΔprfA (lmo0200) [Aké et al., 2011]
E. coli
NM522 supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM) (r – m –) [Gough and Murray, 1983]
k k
[F′proAB lacIqZ ΔGM15]
NM522[pREP4(GroES/GroEL)] supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM) (r – m –) [Amrein et al., 1995]
k k
[F′proAB lacIqZ ΔGM15] (kanR)
Plasmids
pMAD Thermosensitive pE194 replication origin, ermR [Arnaud et al., 2004]
pQE30 His-tag expression vector Qiagen
pREP4(GroES/GroEL) lacI, kanR, produces the chaperones GroES/GroEL [Amrein et al., 1995]
Similar to growth on glycerol, AML73 reproducibly press virulence gene expression [Aké et al., 2011]. It
formed blue colonies when utilizing D -arabitol at con- therefore seems that only efficient PTS-mediated trans-
centrations ranging from 5 to 25 mM . At 20 and 25 mM port of a carbon source can elicit catabolite repression of
D -arabitol, PrfA activity was even stronger than in glyc- L. monocytogenes virulence genes.
erol-grown cells. Repression of the reporter gene was
only observed when the pentitol was used at 50 mM . No
expression of the reporter gene occurred in an AML73-
Experimental Procedures
derived p rfA mutant grown on D -arabitol (online suppl.
fig. S2).
Bacterial Strains, Plasmids, and Growth Conditions
Similar results were obtained when qRT-PCR experi- The L . monocytogenes strains and plasmids used in this study
ments were carried out with RNA isolated from cells are listed in table 2 . Growth studies were carried out at 37 ° C with

shaking in MM [Premaratne et al., 1991] supplemented with the
grown on 25 m M glucose, cellobiose, glycerol, or D -arab-
indicated carbon source in a BioTek microplate reader. Each strain
itol, and primers specific for the virulence gene actA
was grown in duplicate and the absorption at 600 nm (A ) was
600
( fig. 7 ). Almost no actA expression was observed when
measured every 10 min. For qRT-PCR experiments, L. monocyto-
AML73 was grown on cellobiose or glucose. Glycerol- genes cells were grown at 37 ° C with shaking in MM supplemented

grown cells exhibited significant actA expression, which, with the indicated carbon source (25 m M ). For the construction of
deletion or disruption mutants, L. monocytogenes cells were grown
however, was about 4-times higher in D -arabitol-grown
on solid BHI medium at 30 or 42 ° C. To screen L. monocytogenes
AML73. D -Arabitol at concentrations up to 25 mM appar-    
clones during mutant construction, 100 μg/ml 5-bromo-4-chloro-
ently does not repress virulence gene expression, although
3-indolyl β- D -galactopyranoside (X-Gal) was added to solid me-
it is probably catabolized via the PPP. Inhibition of L. dia. Erythromycin was used at a concentration of 5 μg/ml for the
monocytogenes virulence gene expression requires very selection of L. monocytogenes integrants. For fermentation assays
we used the gallery API fermentation kit of bioMérieux (Craponne,
high concentrations of D -arabitol. This resembles the be-
France). Escherichia coli cells were grown in Luria-Bertani medi-
havior of a mutant deleted for the main glucose trans-
um at 37 ° C with shaking. The concentration of antibiotics used for
porter ManL, which takes up the hexose via low-efficien-    
the selection of E. coli transformants was 100 μg/ml for ampicillin
cy PTSs. In this mutant glucose, up to 20 m M did not re- and 30 μg/ml for kanamycin.
Pentitol Utilization by L isteria J Mol Microbiol Biotechnol 2016;26:369–380 377
monocytogenes DOI: 10.1159/000447774
The plasmid pMAD ( table 2 ) contains a thermosensitive pE194 carrying the pQE30-derived plasmid with the lmo2663 gene was
replication origin [Villafane et al., 1987] and produces a thermo- grown at 37 ° C to an OD of 0.5–0.6 before the temperature of
    600
stable β- D -galactosidase from G eobacillus stearothermophilus [Ar- the growth medium was lowered to 30 ° C and 0.1 m M IPTG was

naud et al., 2004]. Plasmid pQE30 (Qiagen) was used for the syn- added. Growth continued at 30 ° C overnight before the cells were

thesis of His-tagged proteins in E. coli strain NM522 or NM522 harvested by centrifugation. Preparation of crude extracts and pro-
containing the plasmid pREP4-GroES/EL [Amrein et al., 1995]. tein purification by ion chelate affinity chromatography were car-
ried out as previously described [Monniot et al., 2012]. Protein
Construction of L. monocytogenes Deletion Mutants concentrations were determined with a dye binding assay [Brad-
To construct EGD-e-derived deletion mutants ( table 2 ), two ford, 1976]. The purity of the eight isolated proteins was deter-
DNA fragments located upstream and downstream from the re- mined by electrophoresis on 0.1% SDS/12.5% polyacrylamide gels
spective gene were amplified by PCR using genomic DNA as the (online suppl. fig. S1). His-tagged ribitol-5-P 2-dehydrogenase
template and the appropriate primer pairs (online suppl. table from L. casei used for several spectrophotometric assays was puri-
S1). In a second PCR, the two obtained amplicons were used as fied as previously described [Bourand et al., 2013].
the template together with the two distal primers, which allowed
fusion of the two DNA fragments. The resulting PCR products Spectrophotometric Enzyme Activity Assays
were digested with the appropriate restriction enzymes and In order to measure the activities of the purified enzymes, we
cloned into the vector pMAD ( table 2 ). For the construction of set up a series of spectrophotometric assays. Because the P-penti-
the deletion mutants Δ lmo2665, Δ lmo2660, Δ lmo0500 , and tols are not commercially available, we measured the presumed
Δ lmo0508, we followed a previously described protocol [Aké et reductase activities of Lmo0506, Lmo2663, and Lmo2664 (EC
al., 2011]. 1.1.1.137; EC 1.1.1.301a; EC 1.1.1.301b) by following the transfor-
mation of D -xylulose-5-P into either D -xylitol-5-P or D -arabitol-
Construction of L. monocytogenes Gene Disruption Mutants 1-P, and of D -ribulose-5-P into either D -ribitol-5-P or D -arabitol-
For the insertional inactivation of the l mo0501 and lmo0503 5-P. We used a 0.5-ml assay mixture containing 50 m M Tris-HCl
genes by single homologous recombination, internal fragments of (pH 7.4), 5 m M MgCl2 , 0.25 m M NADH (Sigma-Aldrich, Saint
about 350 bp of the corresponding genes were amplified by PCR Quentin Fallavier, France), and 0.5 m M D -xylulose-5-P or 0.5 mM
using appropriate primers (online suppl. table S1) and the PCR D -ribulose-5-P (Sigma-Aldrich). After preincubation for 10 min,
products were digested and cloned into pMAD. Disruption of the the reaction was started by adding 20 μg of one of the three penti-
target genes in EGD-e was carried out as previously described tol-P dehydrogenases (Lmo0506, Lmo2663, and Lmo2664). The
[Monniot et al., 2012]. disappearance of NADH was followed by measuring the OD at 340
nm with an UVIKON 9X3W double-beam spectrophotometer
Cloning of D -Arabitol-Related Genes into His Tag Expression (Kontron Bio-Tek) using the ‘Autorate’ program. In order to con-
Vectors firm the dependence of the three pentitol-P dehydrogenases on
The two pentitol operons of L . monocytogenes encode together divalent cations, EDTA (ethylenediaminetetraacetic acid) was
13 catabolic enzymes (f ig. 1 ). Nine of them were amplified by PCR added at a final concentration of 10 mM to the assay mixture, which
using chromosomal DNA of strain EGD-e as the template and an was preincubated for 10 min before the reaction was started. Ow-
appropriate primer pair (online suppl. table S1). The resulting nine ing to the ambiguity about the product formed during ribulose-5-P
PCR products were cut with restriction enzymes and cloned into reduction, we synthesized D -ribitol-5-P by reducing D -ribose-5-P
the His tag expression vector pQE30 (Qiagen). The correct se- with NaBH as previously described [London and Hausman, 1982]
4
quence of each gene was confirmed by DNA sequencing. Eight of and used it as substrate for Lmo2664 and Lmo0506. The assay mix-
the resulting plasmids carrying the different amplified genes were ture contained 50 m M Tris-HCl (pH 7.4), 5 mM MnCl 2 or MgCl2 ,
used to transform E. coli strain NM522. The protein encoded by 0.25 mM NAD (Sigma-Aldrich), and 0.5 mM D -ribitol-5-P.
the lmo2663 gene could not be obtained in soluble form because it Lmo2659 and Lmo0505 transform D -xylulose-5-P (Sigma-Al-
formed inclusion bodies when produced in strain NM522. The drich) into D -ribulose-5-P, and thus function as D -ribulose-5-P
lmo2663 -containing plasmid was therefore used to transform the 3-epimerases (EC 5.1.3.1). The reaction was coupled to the NADH-
E. coli strain NM522[pREP4-GroES/EL], which carries the plas- requiring reduction of D -ribulose-5-P to D -ribitol-5-P by ribitol-
mid pREP4 (Qiagen) containing the genes for the chaperone 5-P 2-dehydrogenase from L. casei [Bourand et al., 2013]. The 0.5-
GroES/GroEL [Amrein et al., 1995]. Overproduction in this strain ml assay mixture contained 50 m M Tris-HCl (pH 7.4), 5 mM
rendered Lmo2663 soluble and allowed its purification. We failed MgCl2 , 0.5 mM NADH, 0.5 mM D -xylulose-5-P, and 18 μg D -ribi-
to purify Lmo0498, which remains insoluble even when produced tol-5-P 2-dehydrogenase (1.3 enzyme units). The reaction was
in NM522[pREP4GroES/EL]. started by adding either Lmo2659 (95 μg) or Lmo0505 (85 μg), and
the disappearance of NADH was monitored by measuring the ab-
Synthesis and Purification of His-Tagged Enzymes sorption at 340 nm.
For the purification of His-tagged proteins from E . coli strain The assay for the presumed D -ribose-5-P isomerase Lmo2662
NM522 carrying plasmid pQE30 containing the different L. mono- (EC 5.3.1.6), which transforms D -ribose-5-P into D -ribulose-5-P,
cytogenes genes, we followed a previously described protocol was also coupled to the reduction of D -ribulose-5-P to D -ribitol-
[Monniot et al., 2012]. The strains were grown at 37 ° C to an opti- 5-P. The 0.5-ml assay mixture contained 50 m M Tris-HCl (pH 7.4),

cal density at 600 nm (OD 600 ) of 0.5–0.6 before 1 mM isopropyl-β- 5 mM MgCl 2 , 0.5 mM NADH, 0.5 mM D -ribose-5-P, and 18 μg (1.3
D -thiogalactopyranoside (IPTG) was added. Growth was contin- U) D -ribitol-5-P 2-dehydrogenase from L . casei . The reaction was
ued for 3 h before the cells were harvested by centrifugation. For started by adding 22 μg of purified Lmo2662 to the assay mixture,
the purification of Lmo2663, strain NM522[pREP4-GroES/EL] and the disappearance of NADH was followed at 340 nm.
378 J Mol Microbiol Biotechnol 2016;26:369–380 Kentache/Milohanic/Cao/Mokhtari/Aké/
DOI: 10.1159/000447774 Ma Pham/Joyet/Deutscher