Downloaded from //aem.asm.org/content/aem/early/2014/10/20/AEM.02484-14.full.pdfOct 20, 2014 ·...
Transcript of Downloaded from //aem.asm.org/content/aem/early/2014/10/20/AEM.02484-14.full.pdfOct 20, 2014 ·...
1
Molecular and metabolic adaptations of Lactococcus lactis at near-zero growth 1
rates 2
3
4
Onur Ercan1,2,3,4, Michiel Wels2,4, Eddy J. Smid2,6, Michiel Kleerebezem2,4,5,* 5
6
Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, The 7
Netherlands1; Top Institute Food and Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, The 8
Netherlands2; Laboratory of Microbiology, Wageningen University, P.O. Box 8033, 6700 EJ 9
Wageningen, The Netherlands3; NIZO food research, P.O. Box 20, 6710 BA Ede, The 10
Netherlands4; Host Microbe Interactomics, Wageningen University, P.O. Box 338, 6700 AH 11
Wageningen, The Netherlands5, Laboratory of Food Microbiology, Wageningen University, P.O. 12
Box 17, 6700 AA Wageningen, The Netherlands6 13
14
Running Head: Molecular adaptations of L. lactis to zero-growth 15
16
Keywords: Lactococcus lactis, zero-growth, transcriptome, amino acids, CodY-binding site, 17
alternative carbon scavenging 18
19
*Address correspondence to Michiel Kleerebezem, Host Microbe Interactomics Group, 20
Wageningen University, De Elst 1, 6708 WD Wageningen, The Netherlands. Phone: +31-317-21
486125; Fax: +31-317-483962; E-mail: [email protected] 22
23
24
25
AEM Accepts, published online ahead of print on 24 October 2014Appl. Environ. Microbiol. doi:10.1128/AEM.02484-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
2
ABSTRACT 26
This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the 27
transition from a growing to a near-zero growth state using carbon-limited retentostat cultivation. 28
Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid 29
fermentation during retentostat cultivation, which appeared to be controlled at the transcription 30
level of the corresponding pyruvate-dissipation encoding genes. During retentostat cultivation, cells 31
continued to consume several amino acids, but also produced specific amino acids, which may 32
derive from the conversion of glycolytic intermediates. We identify a novel motif containing 33
CTGTCAG, in the upstream regions of several genes related to amino acid conversion, which we 34
propose to be the target site for CodY in Lactococcus lactis KF147. Finally, under extremely low 35
carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic 36
functions were found to be highly expressed, which was confirmed by enhanced initial acidification 37
rates on various sugars in cells obtained from near-zero growth cultures. The present integrated 38
transcriptome and metabolite (amino acids and previously reported fermentation end-products) 39
study provides molecular understanding of the adaptation of Lactococcus lactis to conditions 40
supporting low-growth rates, and expands our earlier analysis of the quantitative physiology of this 41
bacterium at near-zero growth rates towards gene regulation patterns involved in zero-growth 42
adaptation. 43
44
45
46
47
48
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
3
INTRODUCTION 49
Fundamental knowledge of microbial physiology and cellular regulation is obtained mainly 50
from studies of microorganisms in batch cultures. However, the pace of life and its associated 51
physiological phases in batch cultivation differ strongly from what is found in natural environments 52
(24). During the early phase of batch cultivations, all nutrients, including carbon and energy sources 53
are usually present in excess, and specific growth rate of the microorganism equals the maximum 54
specific growth rate (4). Thereby, our understanding of microbial energy metabolism originates 55
mostly from microbial population studies performed under laboratory conditions that include rapid 56
growth, high metabolic activity, and high cell density. However, natural microbial communities 57
generally live in relative famine conditions with low specific growth and metabolic rates due to 58
limited supply of nutrients and energy sources (27). Analogously, under specific industrial 59
fermentation conditions, microorganisms may experience strongly restricted access to nutrients for 60
longer periods of time. For example, lactic acid bacteria (LAB) experience long periods of 61
extremely low nutrient availability during the maturing process of dry sausage (25) and cheese (41) 62
productions. Despite these harsh conditions, several LAB succeed to survive in these processes 63
during months of maturation and may continue to contribute to flavor and aroma formation in the 64
product matrix (10, 15, 25). 65
L. lactis is used in food fermentation processes for several products including cheese, sour 66
cream, and other fermented milk products. In these processes, L. lactis converts the available carbon 67
source into lactic acid, resulting in acidification of the food raw-material. In addition, L. lactis is 68
also commonly encountered in diverse natural environments, in particular in decaying plant 69
materials (39). The strain used in this study, L. lactis KF147, was isolated from mung bean sprouts, 70
and its genome sequence reflects many adaptations to the plant-associated habitat, which are in 71
particular apparent from the repertoire of enzymes and pathways predicted to be involved in 72
utilization of plant cell wall polysaccharides. 73
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
4
To study the physiological and genome-wide adaptations of microorganisms to near-zero 74
growth rates, retentostat cultivation or recycling fermenter set-ups have been designed (23). 75
Retentostat cultivation is an adaptation of chemostat cultivation in which a growth-limiting 76
substrate is supplied at a fixed dilution rate, while the complete biomass is retained in the bioreactor 77
by removing the spent medium effluent through an external cross-flow filter. Prolonged retentostat 78
cultivation leads to growth rates that approximate zero while the rate of energy transduction 79
(through substrate consumption and conversion) equals the maintenance energy requirements (e.g., 80
osmoregulation, turnover of damaged cellular components) (14, 47). Therefore, retentostat 81
cultivation comprises a gradual transition from a growing to a near-zero growth state under stable 82
environmental conditions, which sustain high cell-viability. 83
Although retentostat cultivations have been performed to study the fundamental physiology 84
of several microorganisms, including Escherichia coli (7), Bacillus polymyxa (2), Paracoccus 85
denitrificans, Bacillus licheniformis (47), Nitrosomonas europaea and Nitrobacter winogradskyi 86
(43) at low-growth rates, these studies were not consistently complemented with detailed molecular 87
analyses. Exceptions are the retentostat studies performed with Lactobacillus plantarum (22), 88
Aspergillus niger (26), and Saccharomyces cerevisiae (4, 5) that included the analysis of metabolic 89
and transcriptome responses. 90
Previously, we have described how retentostat cultivation allows uncoupling of growth and 91
non-growth-related processes in L. lactis KF147, allowing the investigation of the energy household 92
and quantitative physiology of L. lactis at extremely low-growth rates, exemplified by the estimated 93
specific growth rate of 0.0001 h-1 that corresponds to a doubling time of more than 260 days (14). 94
Despite the imposed extremely low-growth rate, the culture viability was sustained above 90% 95
during prolonged retentostat cultivation (14). This study allowed the accurate calculation of 96
maintenance energy requirements and other quantitative physiology parameters of the strain 97
subjected to retentostat cultivation (14). Furthermore, the calculated values of maintenance-related 98
substrate coefficient (ms) and biomass yield (Ysxmax) revealed that the relative distribution of carbon 99
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
5
source derived energy towards maintenance-associated processes increased from approximately 100
30% to 99% during the transition from growing cultures to prolonged, near zero-growth retentostat 101
cultivation (14). These energy distribution measurements confirmed that the retentostat cultures had 102
reached a typical near-zero growth state after approximately 14 days. Remarkably, the prolonged 103
retentostat culture of L. lactis displayed significant shifts in central carbon metabolism, switching 104
between mixed-acid and lactic-fermentation (14). 105
In the present study, we complement our previous study with an in-depth molecular level 106
analysis of L. lactis KF147 under these retentostat cultivation conditions, including transcriptome, 107
amino acid metabolism and previously obtained fermentation metabolite (14) analyses. Thereby, 108
this study deciphers the molecular adaptation underlying the previously reported physiological 109
observations. The genome-wide transcriptome analyses were in remarkable agreement with the 110
previously observed oscillation of the culture between lactic and mixed-acid fermentation at 111
extremely low-growth rates. Moreover, integrated metabolome and transcriptome analyses created a 112
global view of the interconnected carbon- and nitrogen-metabolism adaptations under these near-113
zero growth conditions. Notably, these adaptations included the progressive relief of carbon 114
catabolite repression, preparing the culture to effectively scavenge alternative carbon sources when 115
they become available. Finally, the transcriptome adaptations are discussed in the context of 116
regulatory circuits that are proposed to govern them, including a predicted role for the central 117
carbon- and nitrogen-metabolism control proteins CcpA and CodY, respectively. 118
MATERIALS AND METHODS 119
Bacterial isolates, media and cultivation conditions. Lactococcus lactis subsp. lactis strain 120
KF147 originates from mung bean sprouts, and its genome sequence was determined (39). Pre-121
cultures for retentostat cultivations (14) were inoculated in 50 ml M17 broth (44) complemented 122
with 0.5% glucose (w/v) and grown overnight at 30°C. Overnight cultures were harvested by 123
centrifugation (6,000 g, 10 min., 4°C) and washed twice with physiological salt solution (0.9% 124
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
6
NaCl in water). Next, the culture was inoculated into chemically defined medium (CDM) 125
containing 0.5% glucose (w/v) for chemostat cultivation. After steady-state had been achieved with 126
six volume changes in chemostat, the fermenters were switched to retentostat mode by withdrawing 127
the effluent through the cross-filter, and pH was controlled at 5.5 through automated 5 M NaOH 128
titration in chemostat and retentostat cultivation (14). To keep the medium composition constant 129
during long-term cultivation, 120-liter batches of medium were prepared, filter sterilized, and used 130
during retentostat cultivations (14). 131
Two independent, carbon source-limited retentostat cultivations were performed under 132
anaerobic conditions, initiating from chemostat cultivation at dilution rates of 0.025 h-1 as 133
previously described (14). Retentostat set-up was assembled with a 1.5-l fermenter (Applikon 134
Biotechnology, Schiedam, The Netherlands) and an autoclavable polyethersulfone cross-flow filter 135
(Spectrum Laboratories, CA, USA). As removal of samples could interrupt biomass accumulation, 136
sample volume and sampling frequency were minimized. 137
Biomass and amino acids determination. During fermentations, culture samples were 138
withdrawn at regular intervals to measure cell dry weight (CDW) and amino acid concentrations. 139
For CDW determination, 5 ml of culture was passed through pre-weighted membrane filters with a 140
pore size 0.45µm (Merck Millipore, Darmstadt, Germany) using a vacuum filtration unit (Sartorius 141
stedim biotech, Gottingen, Germany). Subsequently, membrane filters were dried at 55°C for 24 142
hours, and the biomass collected on the membranes was determined in g/ml. 143
Concentration of amino acids in the culture supernatant and in the medium feed were 144
measured by EZ:fast free amino acid analysis kit (KG0-7165) (Phenomenex, CA, USA). According 145
to the manufacturer’s instructions, the analysis was performed using a gas chromatography (GC) 146
(Thermo Scientific, MA, USA) with a flame ionization detector (FID) (Thermo Scientific, MA, 147
USA). 148
Acidification activity. Acidification profiles were determined in triplicate in 200 µl PBS 149
buffer (initial pH 6) with 0.5% of a chosen carbon source (glucose, ribose, mannitol, sucrose, 150
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
7
fructose, and raffinose) (v/v) at 30°C, inoculated with 5 x 108 cfu/ml of stock cultures of retentostat 151
cultures collected at days 14, 21, 35, and 42, using 96-well microplate HydroPlate® HP96U 152
(PreSens, Regensburg, Germany). These plates encompass an optical pH sensor on the bottom of 153
each well, which can be read-out through the bottom of the plate using a fluorescence reader. 154
According to the manufacturer’s instructions, pH values of cell suspensions in PBS buffers with the 155
chosen carbon source were measured every 10 minutes for 10 hours with a microplate fluorescence 156
reader (Tecan Safire II, Grödig, Austria). The maximum acidification rate (Vmax) value that has an 157
arbitrary and pH-based unit (pH Ux10-3min-1), was calculated on basis of the slope of the pH versus 158
time plot using at least 8 subsequent time-points (regression coefficient > 0.99). 159
RNA isolation and transcriptome analysis. Total L. lactis RNA was isolated from two 160
independent retentostat cultures harvested at days 0, 2, 7, 14, 21, 28, 35, and 42. RNA extraction, 161
reverse transcription, labeling, hybridization, and data analysis were done as previously described 162
(34). Briefly, following methanol quenching, RNA was phenol-chloroform extracted and purified 163
using the High Pure RNA isolation kit (Roche Diagnostics, Mannheim, Germany). Total RNA was 164
used as a template to synthesize cDNA, using the Superscript TMIII reverse transcriptase (RT) 165
enzyme (Invitrogen, Carlsbad, CA), followed by purification by the CyScribe GFX purification kit 166
(GE Healthcare, Buckinghamshire, United Kingdom) and Cy-3 or Cy-5 labeling (Amersham; 167
CyDye postlabeling reactive dye pack; GE Healthcare, Buckinghamshire, United Kingdom). L. 168
lactis KF1471 cDNA was hybridized to OligoWiz (48) designed oligonucleotide DNA microarrays 169
(Agilent Technologies, Santa Clara, CA). After washing (34) slides were scanned at several photo 170
multiplier tube values, and optimal scans were selected on the basis of signal distribution, and data 171
were normalized using the Lowess and inter-slide scaling normalization as available in MicroPrep 172
(46). Median intensities of different probes per gene were taken as absolute gene expression 173
intensities per gene for each condition. The microarray hybridization scheme for the transcriptome 174
analyses at retentostat cultivations consisted of a compound loop design with 26 arrays (Fig. S1). 175
Microarray data and the experimental procedure have been submitted to the NCBI Gene Expression 176
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
8
Omnibus (GEO) under accession number GPL17806 and GSE51494 177
(http://www.ncbi.nlm.nih.gov), respectively. 178
The gene expression intensities were compared and clustered using Short Time-series 179
Expression Minor (STEM) (version 1.3.6, http://www.cs.cmu.edu/~jernst/stem/) (16). The STEM 180
clustering algorithm was used to identify enrichment of Gene Ontology (GO) terms, using 181
Bonferroni correction to determine significance and a maximum number of model profiles of 50. 182
The expressions of genes involved in specific pathways (e.g., glycolysis, pyruvate dissipation, and 183
amino acid metabolism) were projected in heat-maps using the MultiExperiment Viewer (MeV) 184
(http://www.tm4.org/mev/) (37). The correlation of the transcriptome data at each time-point for the 185
two independent retentostat cultivations was calculated by Pearson correlation analysis and 186
displayed as hierarchical clustering using the MeV tool. 187
DNA motif mining. Gene expression data from the significant model profiles identified by 188
STEM were used as data source to identify transcription factor binding sites (TFBSs) in the genome 189
of L. lactis KF147. Binding sites searches were performed using 300 bp upstream regions of each 190
regulated gene using the algorithm for fitting of a mixture model by expectation maximization 191
(MEME) (3), using the parameters mod anr (unlimited number of motifs per upstream sequence), 192
revcomp (allowing motifs to be present on both “+” and “-” strand), and allowing maximally 3 193
motifs to be found in each upstream region without restricting the total number of motifs. The 194
PePPeR database was used as a source of literature based regulon clusters (11). 195
RESULTS 196
Transcriptome data analysis. In our earlier study, metabolic adaptations and physiology of 197
L. lactis KF147 at extreme low-growth rates were studied in anaerobic and carbon-limited 198
retentostat cultivations (sustained for 42 days) (14). During retentostat cultivation biomass 199
accumulated, reaching a plateau level after approximately 14 days and ultimately growth rates 200
declined from 0.025 h-1 to approximately 0.0001 h-1 after 42 days of retentostat cultivation (Fig. 1A; 201
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
9
14). To examine time-resolved transcriptome adaptation of L. lactis KF147 to near- zero growth 202
conditions, samples were taken before starting the retentostat cultivation regime (t = 0 day; 203
chemostat conditions at D = 0.025 h-1), and 2, 7, 14, 21, 28, 35, and 42 days after initiating the 204
retentostat regime, in biologically independent duplicate cultivations. Hierarchical clustering and 205
Pearson correlation analysis illustrated that the transcriptome profiles taken from the two replicates 206
displayed highly similar transcriptome evolutions over time (Pearson correlation > 0.92), supporting 207
the high reproducibility of the experimental set-up (Fig. 1B). In addition, cluster analysis clearly 208
separated transcriptome patterns of samples taken during the growing stages of the experiment 209
(days 0, 2, and 7), from those where growth was stagnating to eventually reach near-zero-growth 210
conditions (days 14, 21, 28, 35, and 42) (Fig. 1B). The discrimination of these two main clusters 211
underpin the separation of the transcriptome signatures related to growth and near-zero growth 212
associated processes. 213
To identify gene expression patterns during the course of the experiment, absolute 214
expression levels of all genes (2533 genes in L. lactis KF147) were subjected to expression cluster 215
analysis using the Short Time-series Expression Minor (STEM) module, which employs a process 216
of statistical clustering of time-series datasets into pre-composed patterns of expression (16). STEM 217
analysis divided the expression patterns into 50 time-resolved model expression profiles, which 218
were sorted on basis of the number of genes assigned to the profile. In total 66.9 % and 64.4 % of 219
the annotated L. lactis KF147 genes in the retentostat duplicates were clustered by STEM into eight 220
and eleven statistically significant model profiles, respectively (Fig. S2A & S2B). Since the 221
congruency between the transcriptome profiles obtained in the replicate experiments was very high, 222
and highly similar STEM profile distributions were obtained for the duplicate retentostat 223
cultivations, the data presented in this study are those obtained from one of the retentostat cultures, 224
which consistently displayed highly congruent expression in the duplicate retentostat cultivation. 225
Since the expression patterns in model profiles 7, 8, 40, and 41 coincided with the carbon 226
metabolism shifts that we described previously (14), we focused on conserved gene sets in these 227
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
10
model profiles (Table 1, Fig. S2 &S4) analyzing the significantly (p-value ≤ 0.05) enriched gene 228
ontology (GO). 229
Cell membrane biosynthesis related processes expression during retentostat cultivation. 230
In STEM-Model profile 8 was enriched for a variety of metabolic processes (Table 1). Genes in this 231
cluster were characterized by progressively reducing expression during the growth associated stages 232
of the retentostat cultivation, followed by a period of stable low expression and subsequent 233
increasing expression during prolonged near-zero growth conditions (Fig. S2 & S3). This cluster 234
also included the category “fatty acid metabolic process” containing the fabDFGHZ and accABCD 235
genes associated with fatty acid biosynthesis (Fig. S4A) of which the expression remained very low 236
also upon prolonged retentostat cultivation. This observation indicates that L. lactis KF147 adapted 237
to near-zero growth by repression of genes related to fatty acid production, thus down regulating the 238
synthesis of one of the main building-blocks of the cell membrane. Model profile 7 clusters genes 239
that were expressed at continuously declining levels during retentostat cultivation (Fig. S2 & S3). In 240
this profile, the overrepresented functional categories related to membrane associated functions 241
(Table 1). These clusters included many ATP-binding cassette (ABC) and phosphotransferase 242
transport systems. In addition, cluster 7 contained genes involved in exopolysaccharide (EPS) 243
synthesis (Fig. S4B & S4C), which is in agreement with the notion that LAB produce and secrete 244
EPSs into their environment only during growth (30). Model profiles 40 and 41 appear to have 245
similar patterns of expression, characterized by initially increasing expression during early stages of 246
retentostat cultivation and remaining stably expressed or slightly declined levels of expression 247
during prolonged retentostat cultivation (Fig. S2 & S3). These model profiles were enriched for 248
functional categories related to RNA synthesis and regulation, transcription and translation (Table 249
1). 250
Central carbon metabolism (CCM) expression during retentostat cultivation. STEM-251
Model profile 8 was enriched for GO terms associated with “organic acid metabolic process” (Table 252
1), and the genes in this class displayed progressively reducing expression during the growth 253
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
11
associated stages of the retentostat cultivation (up to day 7), followed by a period of stable low 254
expression and a recovery of high expression levels during prolonging retentostat conditions and 255
near-zero growth rates (Fig. S2). Thereby, the expression profiles of these genes followed the 256
timing of the carbon and pyruvate dissipation metabolism fluctuations observed during retentostat 257
fermentation (14), which is in good agreement with the “organic acid metabolic process” 258
enrichment. The expression profiles of the genes associated with these processes were visualized 259
using heat-map representation during chemostat (day 0) and retentostat cultivation (Fig. 2). 260
Pyruvate dissipation associated genes displayed a remarkably consistent time-dependent 261
transcription profile during retentostat cultivation. At days 0, 2, and 7, the lactate dehydrogenase 262
encoding gene (ldhL) (involved in lactic acid production) was expressed at a low level, whereas the 263
genes encoding pyruvate dehydrogenase (pdhABCD), alcohol dehydrogenase (adhAE), pyruvate 264
formate lyase (pflA), phosphotransacetylase (eutD), and acetate kinase (ackA1A2), involved in 265
mixed-acid fermentation (production of ethanol, formic acid, and acetate) were highly expressed 266
(Fig. 2B). Subsequently, the transcript level of ldhL was elevated on days 14, 21, and 28, while 267
those of pdhABCD, adhAE, pflA, and eutD, and ackA1A2 were decreased during these stages of 268
cultivation. At the last stage of retentostat cultivation (days 35 and 42), the ldhL gene transcription 269
was again repressed, which coincided with a recovery of the higher expression level of genes 270
involved in mixed-acid fermentation (Fig. 2B). These findings indicate that the fluctuations in 271
pyruvate dissipation behavior during retentostat cultivation (14) accurately reflect the transcriptome 272
profiles of the genes involved in these catabolic pathways, and could explain the mixed-acid and 273
lactic-fermentation switches observed (14). 274
Unlike the regulation of pyruvate-dissipation related genes, the genes of the glycolysis 275
pathway (glk, pgi, pfk, fbaA, tpiA, gapAB, pgk, pmg, enoAB, pyk) were inconsistently regulated in 276
the first week of retentostat cultivation, where all glycolytic genes were repressed or did not change 277
(i.e., < 2-fold change), except the glk, pfk, and fbaA genes (Fig. 2B). However, during subsequent 278
days of retentostat cultivation (days 14 and 21) the expression of glk, pgi, pfk, fbaA, tpiA, gapAB, 279
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
12
pgk, pmg, enoAB, and pyk was consistently up-regulated, whereas their expression was again 280
suppressed during prolonged retentostat cultivation (days 28, 35, and 42), except the gapA, pmg, 281
and enoA genes (Fig. 2B). Moreover, the progressive reduction of the growth rate (Fig. 1A; 14) 282
appears to be paralleled by reduced glycolytic gene expression, although at the time-points that 283
corresponded with increasing lactic acid production, the expression of the glycolytic genes was 284
transiently increased. These findings imply that under retentostat cultivation conditions, the 285
expression pattern of the majority of the glycolytic genes appears to follow the previously described 286
metabolic fluctuations between mixed-acid and lactic-fermentation (14) rather than actual growth 287
rates. The exception appears to be gapA, pmg, and enoA that were continuously expressed at 288
elevated levels after 14 days of retentostat cultivation. Intriguingly, the metabolites associated with 289
these enzymes (substrates and products), also happen to be substrates for the inter-conversion to 290
amino acids biosynthesis, and thereby appear to be consistent with the observed amino acid 291
production at certain stages of the fermentation (see below). 292
Amino acid profiles and amino acid metabolism regulation during retentostat 293
cultivation. Amino acid concentrations were determined (Fig. 3 & S5), at each time-point after 294
initiation of retentostat growth (day 0). The branched chain amino acids (BCAAs) valine, leucine, 295
and isoleucine were constantly consumed during the first week of the retentostat cultivation and 296
subsequently the consumption decreased gradually until days 21 and 28 of the retentostat 297
cultivation, whereas prolonged retentostat cultivation beyond 28 days led to a gradual increase 298
again of the BCAA consumption towards the end of the cultivation (Fig. 3A). Notably, the 299
consumption of the aromatic amino acids (AAAs) phenylalanine, tyrosine, and tryptophan appeared 300
to display similar patterns of concentrations as compared to the BCAAs (Fig. 3B). 301
The GO terms “cellular amino acid metabolic process” and “branched chain family amino 302
acid metabolic process” were overrepresented in transcriptome model profile 8 in both retentostat 303
cultivations (Table 1), and thereby appeared to reflect the pattern of amino acid 304
consumption/production during retentostat cultivation. The ilvABCDH, leuABCD, als, bcaT, and 305
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
13
aspC genes that encode enzymes responsible for BCAAs biosynthesis displayed similar 306
transcription patterns during retentosat cultivation. Their expression fluctuated during the first week 307
of the retentostat cultivation, but consistently decreased after 7 days, followed by increased 308
expression after 35 days of retentostat cultivation (Fig. 4). Notably, genes associated with 309
tryptophan (trpCDFGS) and histidine (hisABDFGHIKZ) syntheses were also clustered in model 310
profile 8 and displayed a similar expression pattern as the BCAA associated genes (Fig. 5). These 311
transcriptional profiles establish a relatively good correspondence between the 312
consumption/production of these amino acids and their biosynthetic pathway encoding genes. 313
The other amino acids appeared to display similar concentration fluctuations as compared to 314
BCAAs and AAAs during retentostat cultivation (Fig. S5). Notably, at certain time-points (days 14 315
and 21) several amino acids appeared to be net-produced by the culture. At these time-points, the 316
genes encoding glutamate synthase, a glycerol-3-phosphate transporter, and a glutamate ABC 317
transporter (gltBDPS), as well as cysteine synthase (cysK), a lysine specific permease (lysQ) and a 318
homoserine kinase and threonine synthase (thrBC) were expressed at low levels, while their 319
expression was higher at stages prior and after the period of net production (Fig. S6). Conversely, 320
the diaminopimelate decarboxylase encoding lysA gene displayed an expression profile that is the 321
inverse of the related lysQ gene (Fig. S6). Taken together, the production of glutamic acid, cysteine, 322
threonine, and lysine coincides with the period that is characterized by enhanced lactic fermentation 323
and could be controlled by the gltBDPS, cysK, thrBC, lysAQ genes, respectively. 324
Identification of a cis-acting DNA-motif potentially involved in near-zero growth gene 325
regulation. Transcriptional regulators strongly control expression levels of genes by binding to 326
TFBSs. To identify candidate DNA-motifs that are potential TFBSs involved in adaptation to near-327
zero growth conditions, we searched for overrepresented DNA sequences in the upstream regions of 328
genes that showed correlated expression. As a result, a highly conserved motif, encompassing the 329
palindromic sequence element 5’-CTGTCAG-3’ (Fig. 6A) was identified in profile 7 and 8. The 330
motif is present upstream of genes related to synthesis of BCAAs (ilvAB, ileS, leuC), histidine 331
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
14
(hisB), cysteine (cysK), arginine (argBF, arcD), serine (serS), tryptophan (trpG), and fatty acid 332
(accD, fabZ); and peptide uptake (dtpT, pepC) (Fig. S7), suggesting that it could play a key role in 333
the adaptation to near-zero growth conditions by its role in nitrogen metabolism regulation of L. 334
lactis KF147 (see discussion). 335
Genome level prediction of enhanced catabolic flexibility of L. lactis KF 147. Model 336
profiles 40 and 41 also encompasses many genes involved in uptake and metabolism of alternative 337
carbon sources such as ribose, mannitol, galacturonate, raffinose, sucrose, and fructose. These genes 338
were relatively lowly expressed during the growth associated stages of retentostat cultivation (up to 339
day 14) and subsequently became gradually higher expressed upon prolonged retentostat 340
cultivation, reaching very high levels of expression during the near-zero growth conditions reached 341
at the end of cultivation (days 35 and 42) (Fig. 7 & S8). For example, the expression of genes 342
associated with uptake and metabolism of ribose (rbsABCDKR) and mannitol (mtlADFR) was 343
increased more than 10-fold during prolonged retentostat cultivation (Fig. 7). These observations 344
illustrate the transcriptional response to prolonged and severe carbon source limitation that is 345
encountered during prolonged retentostat cultivation, leading to the progressive derepression and/or 346
activation of expression of several genes required for the catabolism of alternative carbon sources in 347
L. lactis KF147. These responses raised the question whether these retentostat adapted L. lactis 348
KF147 cultures would display significantly enhanced catabolic flexibility and would more readily 349
and rapidly ferment carbon sources other than glucose. To address this question, the fermentation 350
rate of various carbohydrates by non-growing bacterial suspensions of L. lactis KF147 withdrawn 351
from the retentostat culture on days 14, 21, 35, and 42 were determined. 352
To this end, the maximum acidification rate (Vmax), which was expressed using an arbitrary, 353
pH-based unit (pH Ux10-3min-1; see materials and methods), was determined in L. lactis KF147 354
cell-suspensions derived from the retentostat culture on days 14, 21, 35, and 42 by incubation with 355
glucose, ribose, mannitol, sucrose, fructose, or raffinose as the fermentable substrate. Since the 356
culture biomass did not significantly increase after 14 days of retentostat cultivation and at this 357
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
15
stage the expression level of the relevant genes appeared to be unaffected compared to carbon-358
limited chemostat growth conditions, this sample was used as a reference in these analyses. The 359
highest Vmax values were obtained from the sample taken after 42 days of retentostat cultivation, for 360
all carbon sources used (Table 2). Specifically, the Vmax of the cell suspension derived from the 361
retentostat culture on day 42 was several folds higher than the Vmax of the suspension based on day 362
14 cultures for the carbon sources ribose (3-fold), mannitol (5-fold), sucrose (2-fold), and raffinose 363
(3-fold), respectively (Table 2). Only the acidification rates obtained for fructose and glucose were 364
similar between all time-points evaluated (days 14, 21, 35, and 42), where glucose served as a 365
positive control in these analyses and was consistently associated with the highest acidification rate 366
for all of the suspensions tested (Table 2). These results confirmed that the induced transcription of 367
genes involved in alternative sugar utilization pathways enabled the cells to rapidly adjust to the 368
utilization of alternative carbon sources when these became available. Notably, the progressive 369
derepression of metabolic pathways dedicated to the use of alternative carbon and energy sources 370
clearly exceeds the basal levels of expression that can be seen upon initial carbon limitation 371
conditions, e.g., during carbon-limited chemostat cultivation. 372
DISCUSSION 373
This paper presents the molecular adaptation of L. lactis to near-zero growth rates induced 374
by carbon-limited retentostat cultivation and thereby expands our previous analysis of the 375
quantitative energy household and physiology of this bacterium under these conditions (14). 376
Genome-wide transcriptional data were integrated with metabolite datasets of organic- and amino-377
acid production and consumption under retentostat conditions, allowing the identification of 378
transcription signatures that reflect near-zero growth adaptation inferred by retentostat cultivation. 379
Transcriptome adaptations established the repression of several growth associated functions, 380
specifically those related to the biosynthesis of particular macromolecules, i.e., membrane 381
components and extracellular polysaccharides. Notably the retentostat conditions did not induce 382
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
16
stringency-like responses and genes encoding the components of the machineries for DNA 383
replication, transcription and translation remained relatively highly expressed. 384
Our previous study (14) highlighted that L. lactis KF147 retentostat cultures display 385
intriguing metabolic switches within their central carbohydrate and energy metabolism, fluctuating 386
between mixed-acid and lactic fermentation. Notably, in the present study we show that the 387
fermentation end-product analyses (14) are congruent with the transcriptional patterns of the genes 388
involved in the corresponding pathways, implying that the distribution of pyruvate among the 389
different dissipation pathways is not only controlled by allosteric interactions or redox-balance 390
changes (9, 21), but also controlled at the transcriptional level in L. lactis KF147. Intriguingly, 391
carbon-starved batch cultures of L. lactis strongly suppressed carbon metabolism genes during the 392
stationary phase, but sustained high expression of glycolytic and pyruvate dissipation functions 393
(36). In the current experiments, genes encoding glycolytic enzymes were transiently expressed at 394
elevated levels during early stages of stagnated growth (days 14 to 28), which are proposed to lead 395
to enhanced glycolytic flux that may drive the changes of pyruvate dissipation from mixed-acid 396
towards lactic fermentation, which includes boosted expression of the lactate dehydrogenase. The 397
transcriptional control of glycolytic and pyruvate dissipation pathways appears to be in apparent 398
contradiction with previously proposed regulation by enzyme level control through allosteric 399
interactions with glycolytic intermediates, ATP demand and/or carbon source import rates (8, 21, 400
36, 45). Several glycolytic enzymes have been assigned key roles in the regulation of glycolytic 401
flux in L. lactis as a function of the specific strain of this species and its environmental condition, 402
like the pyruvate kinase and phosphofructokinase in L. lactis MG1363 during batch cultivation on 403
maltose (42, 1) or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in strain NCDO 2118 404
during batch growth on lactose (18). Although the regulation of glycolytic flux in L. lactis is not 405
entirely understood, it has been shown that its flux is not controlled by an individual and rate-406
limiting glycolytic enzyme nor dictated by sugar import rates (33), although the latter have been 407
proposed to predominantly regulate glycolytic flux under conditions of very low carbon flux (8). 408
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
17
Glycolytic flux also appears to be controlled by the cellular energy state, which could elegantly be 409
shown by decreasing ATP levels by increasing F1F0-H+-ATPase expression, which could drive up 410
to 3-fold increased glycolytic flux but led to declining growth rates and biomass yields (28, 29). 411
Nevertheless, mathematical models using the kinetic parameters of the enzymes that constitute the 412
lactococcal glycolytic pathway demonstrated that glycolytic flux could accurately be predicted with 413
such model. This is quite remarkable since the kinetic parameters of enzymes are commonly 414
obtained from in vitro experiments that in many cases neglect many potentially relevant interactions 415
and modifications, including transcriptional regulation, protein phosphorylation, or allosteric 416
modulation (33). The present study revealed that transcriptional regulation contributes to glycolytic 417
flux and pyruvate dissipation control in L. lactis KF147. This could be specific for lactococci 418
isolated from plant origins, like KF147, which would be supported by the many studies that address 419
metabolic control in dairy L. lactis strains that reach alternative but not necessarily mutually 420
exclusive or consistent control-conclusions. Alternatively, this could also be specific for the 421
conditions employed in this study, and thus depend on the specific metabolic characteristics induced 422
by near-zero growth conditions. 423
The response of L. lactis KF147 to extreme low-growth rates also included some remarkable 424
fluctuations of amino acid metabolism. During the continuous reduction of the growth rate in the 425
first retentostat cultivation period, the overall declining rate of amino acid consumption is most 426
likely explained by the reduced requirement for these biomass building-blocks, which has 427
previously also been shown for batch cultures that enter the stationary phase of growth (36). 428
Intriguingly, the transcription of the genes encoding the glycolytic enzymes GAPDH, 429
phosphoglycerate mutase, and enolase which are involved in the production of the intermediates 3-430
phosphoglycerate (3PG), phosphoenolpyruvate (PEP), and pyruvate, were continuously 431
overexpressed after 14 days of retentostat cultivation. These glycolytic intermediates are known to 432
serve as substrates in reactions that link the CCM (central carbon metabolism) to amino acid 433
synthesis, and are involved in the pathways that lead to Ser, Cys, and Gly; Phe, Trp, and Tyr; Ala, 434
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
18
Ile, Leu, Val, and Thr synthesis, respectively (20, 35). This possible role of these intermediates is 435
supported by the observation that intermediate stages (days 14-28) of retentostat cultivation were 436
associated with the net production of certain amino acids and in parallel expressed their glycolytic 437
genes at elevated levels. 438
In several low-GC Gram-positive bacteria, including L. lactis, the transcriptional regulator 439
CodY controls the expression of degradation of oligopeptides, uptake and metabolism of 440
di/tripeptides and amino acids, especially BCAAs, Asn, Glu, His, and Arg, in response to the 441
availability of amino acids or peptides (6, 12). When lactococcal cultures reach the stationary phase, 442
and nutrients become limited, CodY-dependent repression of peptide and amino acid transporter 443
systems is relieved to maintain nitrogen metabolism in the cells (12). Moreover, CodY has also 444
been shown to modulate functions in carbon metabolism since it has been shown to control the 445
expression of citrate synthase (gltA), isocitrate dehydrogenase (icd), and aconitase (citB) that belong 446
to the incomplete Krebs cycle in L. lactis MG1363(12). Importantly, this incomplete Krebs cycle 447
can support the production of α-ketoglutarate, which can serve as a substrate for glutamate 448
production and as a co-substrate for the first step of BCAA catabolism (12, 51), illustrating how 449
CodY regulation could connect nitrogen- to carbon-metabolism regulation (12). Similarly, the 450
detection of a CodY-like regulatory motif in the near-zero growth regulons of L. lactis KF147, led 451
us to propose CodY mediated control of nitrogen metabolism during these conditions, although the 452
expression of the codY gene itself appeared constitutive under the experimental conditions used in 453
this study. This role could include a connecting regulatory role of CodY in CCM regulation via the 454
glycolytic intermediates 3PG, PEP, and pyruvate (see above). Moreover, the connection of pyruvate 455
metabolism and BCAA biosynthesis (Fig. 4A), also implies that CodY could indirectly influence 456
pyruvate dissipation and thereby may play a role in controlling lactic versus mixed-acid 457
fermentation behavior in L. lactis KF147 under retentostat conditions (Fig. 8). 458
In L. lactis MG1363, a 15-bp cis-element with the consensus AATTTTCWGAAAATT has 459
been identified as a high affinity binding site for CodY (12; Fig. 6B). The proposed CodY binding 460
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
19
motif identified in L. lactis KF147 resembles the CodY motif of strain MG1363 (12), despite 461
differences in the deduced consensus sequences for each strain (Fig. 6C). Notably, the motif 462
identified in strain KF147 encompasses a palindromic element and frequently is encountered in a 463
head-to-tail tandem orientation upstream of the identified genes, expanding the palindromic nature 464
of the composite cis-acting element. Therefore, we propose that the motif we identified represents 465
the CodY target site in L. lactis KF147, and that CodY plays a prominent role in the regulation of 466
nitrogen metabolism adaptations in L. lactis KF147 under extremely low-growth rates, and may 467
possibly also control the typical carbon-metabolism fluctuations observed under these conditions in 468
a more indirect manner (Fig. 8). The relevance of nitrogen metabolism regulation in the context of 469
industrial applications of L. lactis is obvious since it has been well established that flavor formation 470
for example in cheese ripening is driven largely by amino acid conversions that involve nitrogen 471
metabolism associated enzymes (40). 472
This study also established that genes involved in import and utilization of alternative 473
carbon sources (other than glucose) were progressively derepressed during prolonged retentostat 474
cultivation, enabling the bacteria to more rapidly switch to alternative energy and carbon resources 475
upon their availability in the environment. This adaptation may reflect the evolutionary benefit of 476
scavenging trace amounts of nutrients in growth limiting environments, which clearly go beyond 477
the relief of catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR; 13, 478
19, 31, 38, 49), since CCR relief was already effective in the carbon-limited chemostat culture that 479
was used to initiate the retentostat cultivation. In LAB, CcpA-mediated CCR includes the 480
repression of catabolic operons in response to the availability of a “preferred” carbon source, which 481
commonly is glucose (19). Intriguingly, in L. lactis CcpA also acts as an activator for the las operon 482
that encodes three key-enzymes in the glycolytic pathway, phosphofructokinase (pfk), pyruvate 483
kinase (pyk) and lactate dehydrogenase (ldh), creating a CcpA connection to central metabolism 484
control (17, 32, 42). The role of CcpA and/or its co-regulators (e.g., HPr, ATP/ADP levels etc.) in 485
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
20
the high level induction of alternative carbon utilization systems during near-zero growth conditions 486
remains to be determined (Fig. 8). 487
In conclusion, transcriptome and metabolite adaptations of L. lactis KF147 to near-zero 488
growth rates inferred by retentostat cultivation are clearly distinct from those elicited by starvation- 489
or stationary phase conditions, and includes particular fluctuating metabolic behavior. The 490
regulation of nitrogen metabolism, and indirectly, possibly also the fluctuating mixed-acid and 491
lactic fermentation patterns, might involve CodY (Fig. 8) through the identified cis-acting motif that 492
resembles the previously reported CodY-box. Retentostat cultivation also led to a progressive relief 493
of carbon catabolite repression and the activation of pathways associated with the utilization of 494
alternative substrates, which goes beyond the canonical CcpA-mediated carbon catabolite 495
regulation. Intriguingly, recent work in Bacillus subtilis established that CcpA and CodY can form a 496
complex that interacts with RpoA, underpinning the interactions between the gene-regulation 497
networks involved in carbon- and nitrogen-metabolism regulation (50). The gene regulation profiles 498
identified in this study include several CcpA and CodY target genes and their regulation may 499
involve a similar regulatory complex encompassing both CodY and CcpA. 500
ACKNOWLEDGEMENTS 501
We thank Sacha van Hijum for designing the hybridization scheme, Jan van Riel for 502
technical assistance with GC, and Marjo Starrenburg for her assistance during hybridization and 503
scanning procedures (NIZO food research, Ede, The Netherlands). In addition, we thank our 504
colleagues from Industrial Microbiology Section, Delft University of Technology and Molecular 505
Genetics Group, University of Groningen in the joint zero-growth project group (Kluyver Centre, 506
Netherlands) for invaluable discussions. 507
This project was carried out within the research programme of the Kluyver Centre for 508
Genomics of Industrial Fermentation which is part of the Netherlands Genomics Initiative / 509
Netherlands Organization for Scientific Research. 510
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
21
REFERENCES 511
1. Andersen, H. W., Solem, C., Hammer, K., and Jensen, P. R. 2001. Twofold reduction of 512
phosphofructokinase activity in Lactococcus lactis results in strong decreases in growth rate and 513
glycolytic flux. J. Bacteriol. 183:3458-3467. 514
2. Arbige, M., and Chesbro, W. R. 1982. Very slow growth of Bacillus polymyxa: stringent 515
response and maintenance energy. Arch. Microbiol. 132:338-344. 516
3. Bailey, T. L., and Elkan, C. 1994. Fitting a mixture model by expextation maximization to 517
discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2:28-36. 518
4. Boender, L. G. M., de Hulster, E. A. F., van Maris, A. J. A., Daran-Lapujade, P., and Pronk, 519
J. T. 2009. Quantitative physiology of Saccharomyces cerevisiae at near-zero specific growth 520
rates. Appl. Environ. Microbiol. 75:5607-5614. 521
5. Boender, L. G. M., van Maris, A. J. A., de Hulster, E. A. F., Almering, M. J. H., van der 522
Klei, I. J., Veenhuis, M., de Winde, J. H., Pronk. J. T., and Daran-Lapujade, P. 2011. 523
Cellular responses of Saccharomyces cerevisiae at near-zero growth rates: transcriptome analysis 524
of anaerobic retentostat cultures. FEMS Yeast Res. 11:603-620. 525
6. Brinsmade, S. R., Kleijn, R. J., Sauer, U., and Sonenshein, A. L. 2010. Regulation of CodY 526
activity through modulation of intracellular branched-chain amino acid pools. J. Bacteriol. 527
192:6357-6368. 528
7. Chesbro, W., Evans, T., and Eifert, R. 1979. Very slow growth of Escherichia coli. J. Bacteriol. 529
139:625-638. 530
8. Cocaign-Bousquet, M. D., Even, S., Lindley, N. D., and Loubiere, P. 2002. Anaerobic sugar 531
catabolism in Lactococcus lactis: genetic regulation and enzyme control over pathway flux. Appl. 532
Microbial. Biotechnol. 60:24-32. 533
9. Collins, L. B. and Thomas, T. D. 1974. Pyruvate kinase of Streptococcus lactis. J. Bacteriol. 534
120:52-58. 535
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
22
10. Crow, V. L., Coolbear, T., Gopal, P. K., Martley, F. G., McKay, L. L., and Riepe, H. 1995. 536
The role of autolysis of lactic acid bacteria in the ripening of cheese. International Dairy Lactic 537
Acid Bacteria Conference 5:855-875. 538
11. de Jong, A, Pietersma, H., Cordes, M., Kuipers, O. P., and Kok, J. 2012. PePPER: A web 539
server for prediction of prokaryote promoter elements and regulons. BMC Genomics 13:299. 540
12. den Hengst, C. D., van Hijum, S. A. F. T., Geurts, J. M. W., Nauta, A., and Kok, J. 2005. 541
The Lactococcus lactis CodY regulon: Identification of a conserved cis-regulatory element. J. 542
Biol. Chem. 280:34332-34342. 543
13. Egli, T. 1995. The ecological and physiological significance of the growth of heterotrophic 544
microorganisms with mixtures of substrates. Adv. Microbial. Ecol. 14:305-386. 545
14. Ercan, O., Smid, E. J., and Kleerebezem, M. 2013. Quantitative physiology of Lactococcus 546
lactis at extreme low-growth rates. Environ. Microbiol. 15:2319-2332. 547
15. Erkus, O., de Jager, V. C. L., Spus, M., van Alen-Boerrigter, I. J., van Rijswijck, I. M. H., 548
Hazelwood, L., Janssen, P. W. M., van Hijum, S. A. F. T, and Kleerebezem, M. 2013. 549
Multifactorial diversity sustains microbial community stability. ISME J. 7:2126-2136. 550
16. Ernst, J. and Bar-Joseph, Z. 2006. STEM: a tool for the analysis of short time series gene 551
expression data. BMC Bioinformatics 7:191. 552
17. Even, S., Lindley, N. D., and Cocaign-Bousquet, M. D. 2001. Molecular physiology of sugar 553
catabolism in Lactococcus lactis IL1403. J. Bacteriol. 183:3817-3824. 554
18. Even, S., Garrigues, C., Loubiere, P., Lindley, N. D., and Cocaign-Bousquet, M. 1999. 555
Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate 556
dehydrogenase activity. Metab. Eng. 1:198-205. 557
19. Fujita, Y. 2009. Carbon catabolite control of the metabolic network in Bacillus subtilis. Biosci. 558
Biotechnol. Biochem. 73:245-259. 559
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
23
20. Ganesan, B., Stuart, M. R., and Weimer, B. C. 2007. Carbohydrate starvation causes a 560
metabolically active but nonculturable state in Lactococcus lactis. Appl. Environ. Microbiol. 561
73:2498-2512. 562
21. Garrigues, C., Loubiere, P., Lindley, N. D., and Cocaign-Bousquet, M. 1997. Control of the 563
shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: Predominant role of 564
the NADH/NAD+ ratio. J. Bacteriol. 179:5282-5287. 565
22. Goffin, P., van de Bunt, B., Giovane, M., Leveau, J. H. J., Höppener-Ogawa, S., Teusink, 566
B., and Hugenholtz, J. 2010. Understanding the physiology of Lactobacillus plantarum at zero 567
growth. Mol. Sys. Bio. 6:413. 568
23. Herbert, D. 1961. A theoretical analysis of continuous culture systems, p. 21-53. In D. W. 569
Hastings et al. (ed.), Continuous culture of microorganisms. Society of Chemical Industry, 570
London, United Kingdom. 571
24. Hoehler, T. M., and Jorgensen, B. B. 2013. Microbial life under extreme energy limitation. 572
Nat. Rev. Microbiol. 11:83-94. 573
25. Hugas, M., and Monfort, J. M. 1997. Bacterial starter cultures for meat fermentation. Food 574
Chem. 59:547-554. 575
26. Jorgensen, T. R., Nitsche, B. M., Lamers, G. E., Arentshorst, M., van den Hondel, C. A., 576
and Ram, A. F. 2010. Transcriptome insights into the physiology of Aspergillus niger 577
approaching a specific growth rate of zero. Appl. Environ. Microbial. 76:5344-5355. 578
27. Koch, A. L. 1971. The adaptive responses of Escherichia coli to a famine and feast existence. 579
Adv. Microb. Physiol. 6:147-217. 580
28. Koebmann, B. J., Solem, C., Pedersen M. B., Nilsson, D., and Jensen P. R. 2002a. 581
Expressing of genes encoding F-1-ATPase result in uncoupling of glycolysis from biomass 582
production in Lactococcus lactis. Appl. Environ. Microbiol. 68:4274-4282. 583
29. Koebmann, B. J., Westerhoff, H. V., Snoep, J. L., Solem, C., Pedersen M. B., Nilsson, D., 584
Michelsen, O., and Jensen, P. R. 2002b. The extent to which ATP demand controls the 585
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
24
glycolytic flux depends strongly on the organism and conditions for growth. Mol. Biol. Rep. 586
29:41-45. 587
30. Laws, A., Gu, Y., and Marshall, V. 2001 Biosynthesis, characterization, and design of bacterial 588
exopolysaccharides from lactic acid bacteria. Biotech. Adv. 19:597-625. 589
31. Lendenmann, U., and Egli, T. 1995 Is Escherichia coli growing in glucose-limited chemostat 590
culture able to utilize other sugars without lag? Microbiology 141:71-78. 591
32. Luesink, E. J., van Herpen, R. E., Grossiord, B. P., Kuipers, O. P., and de Vos, W. M. 1998. 592
Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon 593
in Lactococcus lactis are mediated by the catabolite control protein CcpA. Mol. Microbiol. 594
30:789-798. 595
33. Martinussen, J., Solem, C., Holm, A. K., and Jensen, P. R. 2013. Engineering strategies 596
aimed at control of acidification rate of lactic acid bacteria. Curr. Opinion. Biotech. 24:124-129. 597
34. Meijerink, M., van Hemert, S., Taverne N., Wels, M., de Vos, P., Bron, P. A., Savelkoul, H. 598
F., van Bilsen, J., Kleerebezem, M., and Wells, J. M . 2010. Identification of genetic loci in 599
Lactobacillus plantarum that modulate the immune response of dendritic cells using comparative 600
genome hybridization. PLoS One 5:e10632. 601
35. Novak, L., and Loubiere, P. 2000. The metabolic network of Lactococcus lactis: Distribution 602
of 14C-labeled substrates between catabolic and anabolic pathways. J. Bacteriol. 182:1136-1143. 603
36. Redon, E., Loubiere, P., and Cocaign-Bousquet, M. 2005. Transcriptome analysis of the 604
progressive adaptation of Lactococcus lactis to carbon starvation. J Bacteriol 187:3589-3592. 605
37. Saeed, A. I., Bhagabati, N. K., Braisted, J. C., Liang, W., Sharov, V., Howe, E. A., Li, J., 606
Thiagarajan, M., White, J. A., and Quackenbush, J. 2006. TM4 microarray software suite. 607
Method. Enzymol. 411:134-93. 608
38. Sepers, A. J. B. 1984. The uptake capacity for organic compounds of two heterotrophic 609
bacterial strains at carbon limited growth. Zeitschr. Allg. Mikrobiol. 24:261-267. 610
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
25
39. Siezen, R. J., Bayjanov, J., Renckens, B., Wels, M., van Hijum, S. A. F. T., Molenaar, D., 611
and van Hylckama Vlieg, J. E. T. 2010. Complete genome sequence of Lactococcus lactis 612
subsp. lactis KF147, a plant-associated lactic acid bacterium. J. Bacteriol. 192:2649-2650. 613
40. Smid, E. J., and Kleerebezem, M. 2014. Production of aroma compounds in lactic 614
fermentations. Annu. Rev. Food Sci. Technol. 5:313-326. 615
41. Smit, G., Smit, B. A., and Engels, W. J. M. 2005. Flavour formation by lactic acid bacteria and 616
biochemical flavor profiling of cheese products. FEMS Microbiol. Rev. 29:591-610. 617
42. Solem, C., Koebmann, B., Yang, F., and Jensen, P. R. 2007. The las enzymes control 618
pyruvate metabolism in Lactococcus lactis during growth on maltose. J. Bacteriol. 189:6727-619
6730. 620
43. Tappe, W., Laverman, A., Bohland, M., Braster M., Rittershaus, S., Groeneweg, J., and 621
van Verseveld H. W. 1999. Maintenance energy demand and starvation recovery dynamics of 622
Nitrosomonas europaea and Nitrobacter winogradskyi cultivated in a retentostat with complete 623
biomass retention. Appl. Environ. Microbiol. 65:2471-2477. 624
44. Terzaghi, B. E., and Sandine, W. E. .1975. Improved medium for Lactic Streptococci and their 625
bacteriophages. Appl. Microbiol. 29:807-813. 626
45. Thomas, T. D., Turner, K. W., and Crow, V. L. 1980. Galactose fermentation by 627
Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation. J. Bacteriol. 628
144:672-682. 629
46. van Hijum, S. A. F. T., Garcia de la Nava, J., Trelles, O., Kok, J., and Kuipers, O. P. 2003. 630
MicroPreP: a cDNA microarray data pre-processing framework. Appl. Bioinformatics 2:41-244. 631
47. van Verseveld, H. W., de Hollander, J. A., Frankena, J., Braster, M., Leeuwerik, F. J., and 632
Stouthamer, A. H. 1986. Modeling of microbial substrate conversion, growth and product 633
formation in a recycling fermenter. Antonie Van Leeuwenhoek 52:325–342. 634
48. Wernersson, R., and Nielsen, H. B. 2005. OligoWiz 2.0 – integrating sequence feature 635
annotation into the design of microarray probes. Nucleic Acids Res. 2:2677-2691. 636
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
26
49. Wick, L.M., Quadroni, M., and Egli, T. 2001. Short- and long-term changes in proteome 637
composition and kinetic properties in a culture of Escherichia coli during transition from glucose-638
excess to glucose-limited growth conditions in continuous culture and vice versa. Environ. 639
Microbiol. 3:588-599. 640
50. Wünsche, A., Hammer, E., Bartholomae, M., Völker, U., Burkovski, A., Seidel, G., and 641
Hillen, W. 2012. CcpA forms complexes with CodY and RpoA in Bacillus subtilis. FEBS J. 642
279:2201-2214. 643
51. Yvon, M., Chambellon, E., Bolotin, A., and Roudot-Algaron, F. 2000. Characterization and 644
role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. 645
cremoris NCDO 763. Appl. Environ. Microbiol. 66:571–577 646
647
648
649
650
651
652
653
654
655
656
657
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
27
TABLES 658
Table 1: Gene Ontology (GO) enrichment analysis. This table contains the GO enrichment results 659
for the set of genes shown in the figure S3A and S3B where the enrichment is computed based on 660
actual size enrichment (p-value ≤ 0.05). 661
Model Profile # GO Category Name
7 Membrane
Integral to membrane Membrane part
8
Cellular amino acid metabolic process Fatty acid metabolic process
Organic acid metabolic process Carboxylic acid biosynthetic process
Lipid metabolic process Primary metabolic process
Glutamine family amino acid metabolic process Branched chain family amino acid metabolic process
Arginine metabolic process
40
Sequence-specific DNA binding transcription factor activity RNA biosynthetic process
Regulation of RNA metabolic process Regulation of cellular biosynthetic process
Regulation of gene expression Regulation of macromolecule metabolic process
41
Regulation of transcription, DNA-dependent Carbohydrate kinase activity
Regulation of RNA biosynthetic process Regulation of biological process
662
663
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
28
Table 2: Maximum acidification rates on different carbon source of L. lactis KF147 samples taken 664
on days 14, 21, 35, or 42 of the retentostat cultivation period. 665
Carbon sources Vmax (pH Ux10-3min-1)* 14 21 35 42
Glucose 3.9 ± 0.34 2.7 ± 0.69 3.7 ± 0.41 5.6 ± 0.43 Ribose 0.2 ± 0.05 0.1 ± 0.06 0.5 ± 0.05 0.6 ± 0.06
Mannitol 0.2 ± 0.03 0.2 ± 0.04 0.7 ± 0.03 1.0 ± 0.02 Sucrose 0.5 ± 0.02 0.5 ± 0.01 1.1 ± 0.04 1.1 ± 0.03 Fructose 2.3 ± 0.05 1.8 ± 0.06 2.3 ± 0.04 2.5 ± 0.05 Raffinose 0.3 ± 0.01 0.3 ± 0.01 0.6 ± 0.02 0.9 ± 0.02
* R2, regression coefficient > 0.99 666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
29
FIGURES 682
683
684
685
686
687
688
689
690
691
692
693
694
Fig. 1: Growth of L. lactis KF147 and hierarchical clustering linkage of transcriptome profiles in 695
retentostat culture. A steady-state anaerobic chemostat culture was switched to retentostat mode at 696
time zero. (A) Data points represent average ± mean deviation of measurements of two independent 697
cultures. Specific growth rate (h-1) (diamonds) and biomass accumulation (g.l-1) (squares) of L. 698
lactis KF147 under retentostat conditions (adapted from Ercan, et al., 2013). (B) Hierarchical 699
clustering linkage of retentostat 1 (R1) and 2 (R2) samples. Complete clustering linkage was 700
performed for samples days on 0, 2, 7, 14, 21, 28, 35, and 42 of duplicate retentostat cultivations 701
based on Pearson correlation with using MeV. 702
703
A
B
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
30
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
Fig. 2: Overview of central carbon metabolism in L. lactis KF147. (A) Simple scheme of glycolysis 722
and pyruvate dissipation pathways. One arrow represents one metabolic reaction and dashed line 723
arrows correspond to more than one reaction. Genes are indicated beside the arrows. End-products 724
are indicated in ellipses. (B) Heat-map of L. lactis KF147 glycolysis and pyruvate dissipation genes 725
differentially expressed (on log2-scale, p-value ≤ 0.05) during retentostat cultivation over the 726
beginning of the chemostat (day 0) (retentostat 1). Similar transcriptome results obtained in 727
retentostat 2 confirmed the consistency of these results in an independent experiment. 728
729
Glucose
Fructose-1,6-P
3-P-Glycerate
PyruvateLactate
Acetyl-CoA
Formate
Acetyl-PAcetaldehydeEthanol
Acetate
glk pgi pfk
fbaA tpiA gapAB pgk
pmg enoAB pyk
ldhL
pflA pdhABCD
adhE
adhA
eutD
ackA1A2
Substrate
Product
A
B
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
31
730
731
732
733
734
735
736
737
738
739
740
741
Fig. 3: Concentration of branched chain amino acids (BCAAs) (A) and aromatic amino acids 742
(AAAs) (B) in L. lactis KF147 in retentostat culture. Data points represent average ± mean 743
deviation of measurements of two independent cultures. (A) Concentration of Val (diamonds), Leu 744
(squares), and Ile (triangles). (B) Concentration of Phe (diamonds), Tyr (squares), and Trp 745
(triangles). All concentrations in panels A and B are presented as the difference between the 746
measured concentration in the medium feed and the measured concentration in the filter line efflux 747
samples. Negative numbers indicate net-consumption; positive numbers indicate net-production. 748
749
750
A
B
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
32
751
752
753
754
755
756
757
758
759
760
761
762
763
764
Fig. 4: Overview of branched chain amino acids (BCAAs) biosynthesis in L. lactis KF147. (A) 765
Simple scheme Ile, Val, and Leu amino acid production pathways. One arrow represents one 766
metabolic reaction and dashed line arrows correspond to more than one reaction. Genes are 767
indicated beside the arrows. End-products are indicated in ellipses. (B) Heat-map of L. lactis KF147 768
BCAAs biosynthesis genes differentially expressed (on log2-scale, p-value ≤ 0.05) during 769
retentostat cultivation over the beginning of the chemostat (day 0) (retentostat 1). Similar 770
transcriptome results obtained in retentostat 2 confirmed the consistency of these results in an 771
independent experiment. 772
773
774
775
776
A
B
Pyruvate
(S)-3-Methyl-2-oxopentanoate
Isoleucine
(S)-2-Acetolactate
Valine
(S)-2-Aceto-2-hydroxybutanoate
Leucine
2-Oxoisovalerate
(2S)-2-Isopropylmalate
Acetyl-CoA
(R)-2-Methylmalate
2-OxobutanoateThreonine leuB leuC leuD ilvA
als ilvB ilvH
ilvC ilvD
bcaT
leuA
leuB leuC leuD bcaT
Pyruvate metabolism
aspC
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
33
777 778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
Fig. 5: Overview of Trp (AAA) and His biosynthesis in L. lactis KF147. (A) Simple scheme Trp 797
and His amino acid production pathways. One arrow represents one metabolic reaction and dashed 798
line arrows correspond to more than one reaction. Genes are indicated beside the arrows. End-799
products are indicated in ellipses. (B) Heat-map of L. lactis KF147 BCAAs Trp and His 800
biosynthesis genes differentially expressed (on log2-scale, p-value ≤ 0.05) during retentostat 801
cultivation over the beginning of the chemostat (day 0) (retentostat 1). Similar transcriptome results 802
obtained in retentostat 2 confirmed the consistency of these results in an independent experiment. 803
hisD
hisGZPRPP Phosphoribosyl-
ATP
Phosphoribosyl-AMP
hisI
Phosphoribosyl-formimino-AICAR-P
Phosphoribulosyl-formimino-AICAR-P
Imidazole-glycerol-3P
Imidazole-acetol-P
Histidinol-P
Histidinol
Histidinal
Histidine
Pentose phosphate pathway
hisI
hisB
hisFH
hisA
hisC
hisK
hisD
A
B
Indole
trpB
Glycolysis Chorismate
Anthranilate
trpEG
trpD
N-(5-phospho-D-ribosyl)
anthranilate
1-(2-carboxyphenylamino)-1-deoxy-D-ribulose
5-phosphate
trpF
Indoleglycerol phosphate
trpC
trpAB
trpA
Tryptophan
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
34
804
805
806
807
808
809
810
811
812
813
814
Fig. 6: Weblogo visualization of the postulated CodY motif in L. lactis KF147 (A) and the 815
experimentally identified CodY motif in L. lactis MG1363 (12) (B); alignment of the CodY motifs 816
from both strains. (A) The postulated CodY binding sequence found in L. lactis KF147. The 817
CTGTCAG palindrome sequence that forms the core of the motif is positioned from nucleotide 2 to 818
8. The thymidine at position 5 appears to be conserved as well. (B) The experimentally verified 819
CodY motif in L. lactis MG1363. (C) The consensus CodY motifs identified in L. lactis MG1363 820
and L. lactis KF147, in which the proposed motif sequence is underlined. 821
822
823
824
825
826
827
828
829
Strain Sequence L. lactis KF147 TCTGTCAGTAAATTT (in this study)
L. lactis MG1363 AATTTTCWGAAAATT (12)
B
A
C
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from
35
830
831
832
833
834
835
Fig. 7: Expression graphs of genes involved in (A) ribose and (B) mannitol uptake and metabolism 836
during retentostat cultivation over the beginning of the chemostat (day 0) from model profiles 40 837
and 41 (retentostat 1). Similar transcriptome results obtained in retentostat 2 confirmed the 838
consistency of these results in an independent experiment. 839
840
841
842
843
844
845
846
847
848
849
850
851
852
Fig. 8: Integrated view of adaptive regulation of L. lactis KF147 to near-zero growth rates induced 853
by retentostat cultivation. In rectangular boxes, red and green indicate increased and decreased 854
expression of the mentioned functional categories, respectively. 855
A B
Retentostat mode
Enhanced expression of alternative carbon source systems
Relief of carbon catabolite repression
switch to retentostat mode
Growing state Transition state Near-zero growth state
Nitrogen metabolism control
Glycolytic flux control
Mixed-acid fermentation control
Chemostat
HPr CcpA
CodY
Lactic acid fermentation control
?
7 14 420
Days
on May 15, 2021 by guest
http://aem.asm
.org/D
ownloaded from