BACKGROUND/OBJECTIVESFeeding in infancy is the most significant determinant of the intestinal microbiota in early life. The aim of this study was to determine the gut microbiota of Korean infants and compare the microbiota obtained between breast-fed and formula-fed Korean infants.SUBJECTS/METHODSWe analyzed the microbial communities in fecal samples collected from twenty 4-week old Korean (ten samples in each breast-fed or formula-fed) infants using pyrosequencing.RESULTSThe fecal microbiota of the 4-week-old Korean infants consisted of the three phyla Actinobacteria, Firmicutes, and Proteobacteria. In addition, five species, including Bifidocbacterium longum, Streptococcus salivarius, Strepotococcus lactarius, Streptococcus pseudopneumoniae, and Lactobacillus gasseri were common commensal intestinal microbiota in all infants. The predominant intestinal microbiota in the breast-fed infants (BFI) included the phylum Actinobacteria (average 70.55%), family Bifidobacteriacea (70.12%), genus Bifidobacterium (70.03%) and species Bifidobacterium longum (69.96%). In the microbiota from the formula-fed infants (FFI), the proportion of the phylum Actinobacteria (40.68%) was less, whereas the proportions of Firmicutes (45.38%) and Proteobacteria (13.85%) as well as the diversity of each taxonomic level were greater, compared to those of the BFI. The probiotic species found in the 4-week-old Korean infants were Bifidobacterium longum, Streptococcus salivarius, and Lactobacillus gasseri. These probiotic species accounted for 93.81% of the microbiota from the BFI, while only 63.80% of the microbiota from the FFI. In particular, B. longum was more abundant in BFI (69.96%) than in FFI (34.17%).CONCLUSIONSBreast milk supports the growth of B. longum and inhibits others. To the best of our knowledge, this study was the first attempt to analyze the gut microbiota of healthy Korean infants according to the feeding type using pyrosequencing. Our data can be used as a basis for further studies to investigate the development of intestinal microbiota with aging and disease status.

C4-dicarboxylates and the C4-dicarboxylic amino acid l-aspartate support aerobic and anaerobic growth of Escherichia coli and related bacteria. In aerobic growth, succinate, fumarate, D- and L-malate, L-aspartate, and L-tartrate are metabolized by the citric acid cycle and associated reactions. Because of the interruption of the citric acid cycle under anaerobic conditions, anaerobic metabolism of C4-dicarboxylates depends on fumarate reduction to succinate (fumarate respiration). In some related bacteria (e.g., Klebsiella), utilization of C4-dicarboxylates, such as tartrate, is independent of fumarate respiration and uses a Na+-dependent membrane-bound oxaloacetate decarboxylase. Uptake of the C4-dicarboxylates into the bacteria (and anaerobic export of succinate) is achieved under aerobic and anaerobic conditions by different sets of secondary transporters. Expression of the genes for C4-dicarboxylate metabolism is induced in the presence of external C4-dicarboxylates by the membrane-bound DcuS-DcuR two-component system. Noncommon C4-dicarboxylates like l-tartrate or D-malate are perceived by cytoplasmic one-component sensors/transcriptional regulators. This article describes the pathways of aerobic and anaerobic C4-dicarboxylate metabolism and their regulation. The citric acid cycle, fumarate respiration, and fumarate reductase are covered in other articles and discussed here only in the context of C4-dicarboxylate metabolism. Recent aspects of C4-dicarboxylate metabolism like transport, sensing, and regulation will be treated in more detail. This article is an updated version of an article published in 2004 in EcoSal Plus. The update includes new literature, but, in particular, the sections on the metabolism of noncommon C4-dicarboxylates and their regulation, on the DcuS-DcuR regulatory system, and on succinate production by engineered E. coli are largely revised or new.

Bacteria are able to grow at the expense of both common (succinate, L-malate, fumarate and aspartate) and uncommon (L-tartrate and D-malate) C(4)-dicarboxylates, which are components of central metabolism. Two types of sensors/regulators responding to the C(4)-dicarboxylates function in Escherichia coli, Bacillus, Lactobacillus and related bacteria. The first type represents membrane-integral two-component systems, while the second includes cytoplasmic LysR-type transcriptional regulators. The difference in location and substrate specificity allows the exogenous induction of metabolic genes by common C(4)-dicarboxylates, and endogenous induction by uncommon C(4)-dicarboxylates. The two-component sensors, DcuS and CitA, are composed of an extracellular Per-Arnt-Sim (PAS) domain, two transmembrane helices, a cytoplasmic PAS and the kinase domain. The structures of the extracellular PAS domains of DcuS and CitA have been determined in the ligand-bound and the apo form. Binding of the ligand results in closing and compaction of the binding site, and the structural change gives rise to piston-type movement of the adjacent membrane-spanning helix-2, and signal transmission to the cytoplasmic side. For DcuS, a membrane-embedded construct has been developed that suggests (by experimentation and modeling) that plasticity of the cytoplasmic PAS domain is central to signal transduction from the membrane to the kinase. Sensor kinase DcuS of E. coli requires the C(4)-dicarboxylate transporters DctA or DcuB as co-sensors for function under aerobic and anaerobic conditions, respectively. DcuB contains a regulatory site that controls the function of DcuS and is independent from the transport region. Therefore, DcuS senses C(4)-dicarboxylates in two independent modes, responding to the effector concentration and the metabolic flux of extracellular C(4)-dicarboxylates.

AcrR, the toxic-compounds-response regulator, regulates motility in microorganisms, presumably to escape from toxic environments. In this study, the genome-wide target genes of AcrR were investigated in a Δ mutant strain by microarray analysis. In the absence of AcrR, the transcription of most flagella/motility genes was highly increased. In addition, flagella formation was increased in this mutant strain. Motility assays revealed that AcrR modulates swimming motility, but not swarming.

Quintuple mutants of Escherichia coli deficient in the C(4)-dicarboxylate carriers of aerobic and anaerobic metabolism (DctA, DcuA, DcuB, DcuC, and the DcuC homolog DcuD, or the citrate/succinate antiporter CitT) showed only poor growth on succinate (or other C(4)-dicarboxylates) under oxic conditions. At acidic pH (pH 6) the mutants regained aerobic growth on succinate, but not on fumarate. Succinate uptake by the mutants could not be saturated at physiological succinate concentrations (< or =5 mM), in contrast to the wild-type, which had a K(m) for succinate of 50 microM and a V(max) of 35 U/g dry weight at pH 6. At high substrate concentrations, the mutants showed transport activities (32 U/g dry weight) comparable to that of the wild-type. In the wild-type using DctA as the carrier, succinate uptake had a pH optimum of 6, whereas succinate uptake in the mutants was maximal at pH 5. In the mutants succinate uptake was inhibited competitively by monocarboxylic acids. Diffusion of succinate or fumarate across phospholipid membranes (liposomes) was orders of magnitude slower than the transport in the wild-type or the mutants. The data suggest that mutants deficient in DctA, DcuA, DcuB, DcuC, DcuD (or CitT) contain a carrier, possibly a monocarboxylate carrier, which is able to transport succinate, but not fumarate, at acidic pH, when succinate is present as a monoanion. Succinate uptake by this carrier was inhibited by addition of an uncoupler. Growth by fumarate respiration (requiring fumarate/succinate antiport) was also lost in the quintuple mutants, and growth was not restored at pH 6. In contrast, the efflux of succinate produced during glucose fermentation was not affected in the mutants, demonstrating that, for succinate efflux, a carrier different from, or in addition to, the known Dcu and CitT carriers is used.