Citrate metabolism and aroma compound production in lactic acid bacteria

24 p.-8 fig.-1 tab.The main activity of lactic acid bacteria (LAB) during fermentation is the catabolism of sugars present in food, producing lactic acid by homoor heterofermentative pathways. In addition, these microorganisms also have the capability to metabolize other substrates, such as citrate. Citrate is present in fruit juices, milk and vegetables and is also added as a preservative to foods. Citrate fermentation by LAB leads to the production of 4-carbon compounds, mainly diacetyl, acetoin and butanediol, which have aromatic properties. One of these compounds, diacetyl is responsible for the buttery aroma of dairy products such as butter, acid cream and cottage cheese. In addition, it is an important component of the flavour of different kinds of chesses and yoghurt. Moreover, the CO2 produced as a consequence of citrate metabolism contributes to the formation of "eyes" (holes) in Gouda, Danbo and other cheeses. Thus, the utilization of citrate in milk by LAB has a very positive effect on the quality of the end products. Therefore, the interest of the dairy industry in controlling citrate utilization by LAB has promoted research into the proteins and effectors controlling its metabolic pathway. In this chapter we summarize the current knowledge of citrate utilization by LAB. The transport of citrate and its metabolism to pyruvate, as well as further conversion to aroma compounds, is described and, the differences in the co-metabolism of citrate with glucose between homo or heterofermentative bacteria is discussed. In addition, the molecular mechanisms controlling expression of genes responsible for transport and conversion of citrate into pyruvate are presented, as are their correlation with the physiological function of citrate metabolism. To date, two different models of regulation have been described which are unique to LAB. In Lactococcus lactis, a specific transcriptional activation of the promoters controlling the cit operons takes place at low pH to provide an adaptative response to acidic stress. In Weissella paramesenteroides, the CitI transcriptional regulator functions as a citrate-activated switch allowing the cell to optimize the generation of metabolic energy. CitI, its operators and citrate transport and metabolic operons are highly conserved in several LAB. Therefore, this mechanism of sensing and response to citrate appears to have been conserved and propogated during the evolution of LABThis work was supported by the European Union grants QLK1-CT-2002-02388and KBBE-CT-2007-211441, the Spanish Ministry of Education grant AGL2006-1193-C05-01 and the Agencia Nacional de Promoción Científica y Tecnológica grant PICT 15-38025Peer reviewedPostprin

Catabolite repression of the citST two-component system in Bacillus subtilis

In Bacillus subtilis, expression of the citrate transporter CitM is under strict control. Transcription of the citM gene is induced by citrate in the medium mediated by the CitS–CitT two-component system and repressed by rapidly degraded carbon sources mediated by carbon catabolite repression (CCR). In this study, we demonstrate that citST genes are part of a bicistronic operon. The promoter region was localized in a stretch of 58 base pairs upstream of the citS gene by deletion experiments. Transcription of the operon was repressed in the presence of glucose by the general transcription factor CcpA. A distal consensus cre site in the citS-coding sequence was implicated in the mechanism of repression. Furthermore, this repression was relieved in Bacillus subtilis mutants deficient in CcpA or Hpr/Crh, components essential to CCR. Thus, we demonstrate that CCR represses the expression of the citST operon, which is responsible for the induction of citM, through the cre site located 1326 bp from transcriptional start site of citST.

Acinetobacter baumannii NCIMB8209: a Rare Environmental Strain Displaying Extensive Insertion Sequence-Mediated Genome Remodeling Resulting in the Loss of Exposed Cell Structures and Defensive Mechanisms

ABSTRACT Acinetobacter baumannii represents nowadays an important nosocomial pathogen of poorly defined reservoirs outside the clinical setting. Here, we conducted whole-genome sequencing analysis of the Acinetobacter sp. NCIMB8209 collection strain, isolated in 1943 from the aerobic degradation (retting) of desert guayule shrubs. Strain NCIMB8209 contained a 3.75-Mb chromosome and a plasmid of 134 kb. Phylogenetic analysis based on core genes indicated NCIMB8209 affiliation to A. baumannii, a result supported by the identification of a chromosomal blaOXA-51-like gene. Seven genomic islands lacking antimicrobial resistance determinants, 5 regions encompassing phage-related genes, and notably, 93 insertion sequences (IS) were found in this genome. NCIMB8209 harbors most genes linked to persistence and virulence described in contemporary A. baumannii clinical strains, but many of the genes encoding components of surface structures are interrupted by IS. Moreover, defense genetic islands against biological aggressors such as type 6 secretion systems or CRISPR-cas are absent from this genome. These findings correlate with a low capacity of NCIMB8209 to form biofilm and pellicle, low motility on semisolid medium, and low virulence toward Galleria mellonella and Caenorhabditis elegans. Searching for catabolic genes and concomitant metabolic assays revealed the ability of NCIMB8209 to grow on a wide range of substances produced by plants, including aromatic acids and defense compounds against external aggressors. All the above features strongly suggest that NCIMB8209 has evolved specific adaptive features to a particular environmental niche. Moreover, they also revealed that the remarkable genetic plasticity identified in contemporary A. baumannii clinical strains represents an intrinsic characteristic of the species. IMPORTANCE Acinetobacter baumannii is an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) opportunistic pathogen, with poorly defined natural habitats/reservoirs outside the clinical setting. A. baumannii arose from the Acinetobacter calcoaceticus-A. baumannii complex as the result of a population bottleneck, followed by a recent population expansion from a few clinically relevant clones endowed with an arsenal of resistance and virulence genes. Still, the identification of virulence traits and the evolutionary paths leading to a pathogenic lifestyle has remained elusive, and thus, the study of nonclinical (“environmental”) A. baumannii isolates is necessary. We conducted here comparative genomic and virulence studies on A. baumannii NCMBI8209 isolated in 1943 from the microbiota responsible for the decomposition of guayule, and therefore well differentiated both temporally and epidemiologically from the multidrug-resistant strains that are predominant nowadays. Our work provides insights on the adaptive strategies used by A. baumannii to escape from host defenses and may help the adoption of measures aimed to limit its further dissemination

Enterococcus faecalis Uses a Phosphotransferase System permease and a host colonization-related ABC transporter for maltodextrin uptake

Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of alpha-1,4- and some alpha-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6 ''-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6 ''-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection. IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two differen

Differential Role of the T6SS in Acinetobacter baumannii Virulence.

Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner

Enterococcus faecalis Uses a Phosphotransferase System permease and a host colonization-related ABC transporter for maltodextrin uptake

Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of alpha-1,4- and some alpha-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6 ''-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6 ''-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection. IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two differen

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6-phosphate phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E.faecalis lacks a malA gene encoding a 6-phospho--glucosidase, which in B.subtilis hydrolyses maltose 6-P into glucose and glucose 6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6-P into glucose 1-P and glucose 6-P. However, purified MalP phosphorolyses maltose but not maltose 6-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B.subtilis mutant accumulating maltose 6-P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6-P, and the resulting maltose is converted by the B.subtilis maltose phosphorylase into glucose and glucose 1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalysed metabolism. Dephosphorylation assays with a wide variety of phospho-substrates revealed that MapP preferably dephosphorylates disaccharides containing an O--glycosyl linkage

The <i>A</i>. <i>baumannii</i> DSM30011 T6SS is required for out-competing <i>E</i>. <i>coli</i>.

<p>A) Representative image showing survival of DH5α <i>E</i>. <i>coli</i> after incubation in growth medium (control) or with <i>A</i>. <i>baumannii</i> ATCC17978 wild type and Δ<i>tssM</i> strains at a 1:1 ratio with or without the presence of a membrane (+M) and B) corresponding quantification (<i>N</i> = 6). C) Survival of DH5α <i>E</i>. <i>coli</i> after incubation in growth medium (control) or with <i>A</i>. <i>baumannii</i> DSM30011 wild type, Δ<i>tssM</i> and Δ<i>tssM</i> complemented strains at a 1:1 ratio, with D) corresponding quantification (<i>N</i> = 4). E) 18% SDS-PAGE (stained with Coomassie Blue) analysis of Hcp secretion in concentrated culture supernatants of <i>A</i>. <i>baumannii</i> DSM30011 wild type, Δ<i>tssM</i> and <i>tssM</i> complemented strains grown up to exponential phase in TSB. Molecular markers are indicated on the left. All quantifications are expressed as means ± SDM plotted in a logarithmic scale.</p

Ability of different <i>A</i>. <i>baumannii</i> strains to out-compete <i>E</i>. <i>coli</i>.

<p>A) Comparative analysis between the T6SS gene loci of <i>A</i>. <i>baumannii</i> ATCC17978, DSM30011 and clinical strains. Black arrows indicate genes specific to <i>Acinetobacter</i>. B) Survival of DH5α <i>E</i>. <i>coli</i> after incubation in growth medium (control) or with <i>A</i>. <i>baumannii</i> ATCC17978 or DSM30011 at the indicated <i>A</i>. <i>baumannii</i>: <i>E</i>. <i>coli</i> ratios (top panel). Bottom panel corresponds to survival of apramycin-resistant DH5α <i>E</i>. <i>coli</i> after incubation in growth medium (control) or with Ab242, Ab244 or Ab825 strains at the indicated ratios. Images correspond to one representative experiment from a minimum of three independent assays quantified in C) to visualize experimental variation, expressed as means ± SDM plotted in a logarithmic scale.</p