Global transcriptome response in Lactobacillus sakei during growth on ribose (original) (raw)
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Adaptation of Lactobacillus sakei to meat: a new regulatory mechanism of ribose utilization?
Le Lait, 2001
Lactobacillus sakei is a lactic acid bacterium commonly found on meat and meat products. Meat is a rich substrate but contains small amounts of sugars, mainly glucose and ribose. The phosphotransferase system (PTS) is a multienzymatic complex responsible for sugar uptake. It is also involved in the regulation of metabolism through various mechanisms (catabolite activation and repression, inducer expulsion and exclusion). The ptsHI operon of L. sakei, encoding the general enzymes of the PTS, was studied and mutants were constructed. On ribose, these mutants grow twice as fast as the wild-type strain. This phenotype was never described in other bacteria and suggests that the PTS regulates ribose utilization. When compared to what is known from the regulations involving the PTS in other bacteria, this mechanism might be new. In Bacillus subtilis and Escherichia coli, in which ribose catabolism was investigated, ribose is transported by an ABC transporter, encoded by rbsABCD genes and then phosphorylated by the rbsK encoded ribose kinase. Ribose-5P is then metabolized through the pentose-P pathway involving xylulose-5P phosphoketolase and acetate kinase. Whereas phosphoketolase and acetate kinase activities remained unchanged in L. sakei ptsI mutants, ribose kinase activity and uptake were increased by a factor of 2.5 and 1.5, respectively. The target of the PTS regulation would thus be transport and/or phosphorylation of ribose. The gene cluster encoding a ribose transporter, ribose kinase and a regulator was cloned and sequenced. In L. sakei no gene encoding RbsA, RbsB or RbsC could be found. However, rbsD was present as well as a new gene rbsU, encoding a protein homologous to a glucose transporter responsible for facilitated diffusion of glucose. The rbsUDK operon is induced by ribose via the regulator RbsR encoded by rbsR located downstream of rbsUDK. In ptsI mutants, this operon was not overexpressed on ribose. This shows that the regulation of ribose utilization is not a transcriptional regulation. Upstream from the rbs operon, a gene encoding acetate kinase (ackA) was found. In other bacteria in which these genes were identified, ackA and the rbs operon are not linked. Moreover, in B. subtilis, ackA is regulated by catabolite activation whereas the rbs operon is repressed by catabolite repression, two mechanisms involving the PTS. In L. sakei, ackA and the rbs operon are adjacent on the
Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis
BMC Microbiology, 2010
Background: Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish.
Journal of molecular microbiology and biotechnology, 1999
A 7-kb DNA fragment of Lactobacillus sakei, containing the rbsD, rbsK and rbsR genes was sequenced. The genes responsible for ribose utilization are organized differently from what was previously described for model organisms such as Escherichia coli and Bacillus subtilis. No gene encoding RbsA, RbsB and RbsC, the subunits of the ribose ABC-transporter, were present in the rbs gene cluster. Instead, we found an open reading frame coding for RbsU, a protein similar to GltA, the glucose transporter of Staphylococcus xylosus. The disruption of rbsK, encoding the ribokinase, impaired growth on ribose. The disruption of rbsR, encoding the repressor, had no effect on the ability to grow on ribose, but led to overexpression of a large transcript corresponding to rbsU, rbsD and rbsK, suggesting that RbsU might be involved in ribose utilization. Ribose uptake and phosphorylation assays on the wild type strain and various mutants showed that, in ptsI mutants, both ribose uptake and phosphoryl...
PloS one, 2017
Effects of glucose availability were investigated in Lactobacillus sakei strains 23K and LS25 cultivated in anaerobic, glucose-limited chemostats set at high (D = 0.357 h-1) and low (D = 0.045 h-1) dilution rates. We observed for both strains a shift from homolactic towards more mixed acid fermentation when comparing high to low growth rates. However, this change was more pronounced for LS25 than for 23K, where dominating products were lactate>formate>acetate≥ethanol at both conditions. A multivariate approach was used for analyzing proteome and transcriptome data from the bacterial cultures, where the predictive power of the omics data was used for identifying features that can explain the differences in the end-product profiles. We show that the different degree of response to the same energy restriction revealed interesting strain specific regulation. An elevated formate production level during slow growth, more for LS25 than for 23K, was clearly reflected in correlating py...
Molecular Physiology of Sugar Catabolism in Lactococcus lactis IL1403
Journal of Bacteriology, 2001
The metabolic characteristics of Lactococcus lactis IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.
Molecular Microbiology, 2002
We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate–carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single-transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I-deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser-46 is assumed to be important for the regulation of sugar metabolism in Gram-positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser-46 was either prevented or diminished (replacement of Ser-46 of HPr with Ala or Thr respectively). In a third mutant, Ile-47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P–Ser–HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non-PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser-46–Ala or Ile-47–Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P–Ser–HPr/CcpA-mediated mechanism of CCR is common to most low G+C Gram-positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P–Ser–HPr participates also in inducer exclusion.
BMC genomics, 2016
Lactobacillus oligofermentans has been mostly isolated from cold-stored packaged meat products in connection with their spoilage, but its precise role in meat spoilage is unknown. It belongs to the L. vaccinostercus group of obligate heterofermentative lactobacilli that generally ferment pentoses (e.g. xylose and ribose) more efficiently than hexoses (e.g. glucose). However, more efficient hexose utilization can be induced. The regulation mechanisms of the carbohydrate catabolism in such bacteria have been scarcely studied. To address this question, we provided the complete genome sequence of L. oligofermentans LMG 22743(T) and generated time course transcriptomes during its growth on glucose, ribose and xylose. The genome was manually annotated and its main functional features were examined. L. oligofermentans was confirmed to be able to efficiently utilize several hexoses and maltose, which is, presumably, induced by its repeated cultivation with glucose in vitro. Unexpectedly, in...
Molecular cloning and analysis of the ptsHI operon in Lactobacillus sake
Applied and environmental microbiology, 1997
The ptsH and ptsI genes of Lactobacillus sake, encoding the general enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), were cloned and sequenced. HPr (88 amino acids), encoded by ptsH, and enzyme I (574 amino acids), encoded by ptsI, are homologous to the corresponding known enzymes of other bacteria. Nucleotide sequence and mRNA analysis showed that the two genes are cotranscribed in a large transcript encoding both HPr and enzyme I. The transcription of ptsHI was shown to be independent of the carbon source. Four ptsI mutants were constructed by single-crossover recombination. For all mutants, growth on PTS carbohydrates was abolished. Surprisingly, the growth rates of mutants on ribose and arabinose, two carbohydrates which are not transported by the PTS, were accelerated. This unexpected phenotype suggests that the PTS negatively controls ribose and arabinose utilization in L. sake by a mechanism different from the regulation involving HPr describe...
BMC Genomics, 2015
The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale.
Microbiology-sgm, 2006
Lactobacillus casei transports glucose preferentially by a mannose-class phosphoenolpyruvate : sugar phosphotransferase system (PTS). The genomic analysis of L. casei allowed the authors to find a gene cluster (manLMNO) encoding the IIAB (manL), IIC (manM) and IID (manN) proteins of a mannose-class PTS, and a putative 121 aa protein of unknown function (encoded by manO), homologues of which are also present in man clusters that encode glucose/mannose transporters in other Gram-positive bacteria. The L. casei man operon is constitutively expressed into a manLMNO messenger, but an additional manO transcript was also detected. Upstream of the man operon, two genes (upsR and upsA) were found which encode proteins resembling a transcriptional regulator and a membrane protein, respectively. Disruption of either upsR or upsA did not affect manLMNO transcription, and had no effect on glucose uptake. Cells carrying a manO deletion transported glucose at a rate similar to that of the wild-type strain. By contrast, a manM disruption resulted in cells unable to transport glucose by the PTS, thus confirming the functional role of the man genes. In addition, the manM mutant exhibited neither inducer exclusion of maltose nor glucose repression. This result confirms the need for glucose transport through the PTS to trigger these regulatory processes in L. casei.