Insights From Genomics

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Nowadays more and more bacteria are undergoing genome sequencing and as a result over 130 completed bacterial genomes have become available in the public domain. Following the first example of Haemophilus influenzae in 1995 (50) the major focus of these efforts has initially been on pathogenic bacteria and includes the completion of several genome sequences of food-borne pathogens, including Bacillus cereus (51), Salmonella typhimurium (52), and Listeria monocytogenes (53). Over the last years sequencing of the genomes of food-associated, non-pathogenic bacteria has received considerable attention, including the elucidation of the complete genome sequence of Bacillus subtilis in 1997 (54). Moreover, the first complete LAB genome sequence published was that of Lactococcus lactis subspecies lactis strain IL1403 (55). To date, only two other high-fidelity genome sequences of LAB, L. plantarum strain WCFS1 (56) and L. johnsonii strain NCC533 (57), have been published. An additional number of LAB genomes is nearing completion and draft genome information has become available in the public domain in 2002 with the publication and appearance of genome sequences for LAB provided by the Joint Genome Institute (http://genome.jgi-psf.org/microbial/) in collaboration with the lactic acid bacteria genomics consortium (58,59). Next to this large amount of sequence data from food-associated LAB, successful efforts have been put in determination of the (complete) genome sequences of members of our normal colonic microbiota, in particular Bacteroides thetaiotaomicron (60) and Bifidobacterium longum (Fig. 4) (61).

L. plantarum is a versatile and flexible organism that is able to grow on a wide variety of sugar sources. This phenotypic trait is reflected in the genome sequence of L. plantarum, which harbours a remarkably high number of 25 complete PTS enzyme II complexes as well as several incomplete complexes. This high number of PTS systems is far more than that found in other complete bacterial genomes, and similar only to Listeria monocytogenes (53) and Enterococcus faecalis (62). In addition to these PTS systems, the L. plantarum genome encodes 30 transporters that are predicted to be involved in the transport of carbon sources. This high sugar uptake flexibility has also been observed in the genomes of other LAB, such as L. johnsonii (57) and L. acidophilus (http://www.calpoly.edu/~rcano/Lacto_genome. html). Moreover, a remarkably high percentage of regulatory genes (8.5%) appeared to be

~ Escherichia coli (4.6)

Bifidobacterium breve (2.4)

Bifidobacterium longum (2.3)

Propionibacteriumfreudenreichii (2.6) ~Brevibacterium linens (3.0)

Pediococcus pentosaceus (2.0) Lactobacillus rhamnosus (2.4) Lactobacillus casei (2.6) Lactobacillus sakeiC\ .9) ' ' I bacillus plantarum (3.3)

•Lactobacillus johnsonii (2.0)

Lactobacillus qasseri (1.8) 'Lactobacillus delbrueckii (2.3) 'Lactobacillus acidophilus (2.0)

Lactobacillus helveticus (2.4) -Enterococcus faecalis (3.2)

■Bacillus subtilis (4.2) Bacillus cereus (4.2)

-Bacillus anthracis (5.2)

-Listeria monocytogenes (2.9)

-Lactococcus lactis (2.3)

'Streptococcus thermophilus (1.8) "Oenococcus oeni (1.8) Leuconostoc mesenteroides (2.0) ■Clostridium perfringens (3.0)

Bacteroides thetaiotaomicron (6.3)

Figure 4 Phylogenetic relationship based upon the neighbor-joining method of partial 16S rDNA sequences (Escherichia coli positions 107 to 1434). It should be noted that for some species the genome sequence has (partially) been determined for multiple strains. LAB genomes are underscored, and published, complete genomes are shown in bold. The estimated genome sizes are indicated between brackets.

encoded in the L. plantarum genome. Similar percentages were found in Listeria monocytogenes, in which 7.3% of all the encoded genes were predicted to be involved in regulatory functions. This could be a reflection of the many different environmental conditions that these bacteria face. Moreover, these sophisticated regulatory systems enable these organisms to adapt quickly to changes in the sugar composition of the host's diet during residence in the proximal parts of the GI tract (Fig. 5).

Ingestion of carbohydrates by the host; high diet-dependent variation

Figure 5 Molecular model of bacterial sugar utilization in the GI tract. In the small intestine mono- and disaccharides are rapidly consumed by the host. Typically, bacteria that live in this niche display highly flexible sugar utilization capacities, allowing them to quickly adapt to changes in the carbon source availability that is determined by the host's diet. This high sugar flexibility is required to compete with the host for carbon acquisition. In the large intestine more complex oligo- and polysaccharides are the only available C-source. Therefore, bacteria in this niche are usually able to hydrolyse complex dietary polysaccharides and host-derived glycoproteins and glycoconjugates. Subsequently, the released, simpler sugars are utilized as C-source by the host and the bacteria residing in the colon. Source: From Ref. 63.

Figure 5 Molecular model of bacterial sugar utilization in the GI tract. In the small intestine mono- and disaccharides are rapidly consumed by the host. Typically, bacteria that live in this niche display highly flexible sugar utilization capacities, allowing them to quickly adapt to changes in the carbon source availability that is determined by the host's diet. This high sugar flexibility is required to compete with the host for carbon acquisition. In the large intestine more complex oligo- and polysaccharides are the only available C-source. Therefore, bacteria in this niche are usually able to hydrolyse complex dietary polysaccharides and host-derived glycoproteins and glycoconjugates. Subsequently, the released, simpler sugars are utilized as C-source by the host and the bacteria residing in the colon. Source: From Ref. 63.

The genomes of B. thetaiotaomicron and Bifidobacterium longum encode an elaborate apparatus for acquiring and hydrolysing otherwise indigestible dietary polysaccharides (60,61). In B. thetaiotaomicron this "colonic substrate dependence" is associated with an environment-sensing system consisting of a large repertoire of extracytoplasmic function sigma factors and one- and two-component signal transduction systems (60). In contrast, genes involved in sugar transport and hydrolysis in Bifidobacterium longum are organized in operons which are predominantly regulated by LacI-type, sugar responsive repressors (61). The tight regulation of sugar utilization observed in these bacteria allows a stringent response to environmental changes and is in accordance with the fact that Bifidobacterium longum and B. thetaiotaomicron need to adapt to wide fluctuations in substrate availability in the colon (60,61). It is speculated that the mode of regulation via repression of genes could allow a quicker response in Bifidobacterium longum (61). Similarly, an operon in L. acidophilus involved in utilization of the prebiotic compound fructooligosaccharide contains a LacI type repressor. Moreover, the expression of this operon is subject to global catabolite repression in the presence of readily fermentable sugars (64). Another interesting finding in the genome of B. thetaiotaomicron is that it appears to encode the capacity to use a variety of host-derived glycoproteins and glycoconjugates. Sixty-one percent of its glycosylhydrolases are predicted to be located in the periplasm, outer membrane, or extracellularly. This suggests that these enzymes are not only important for fulfilling the needs of B. thetaiotaomicron but may also help shape the metabolic milieu of the intestinal ecosystem in ways conducive to maintaining a microbiota that supplies the host with 10 to 15% of our daily calories as fermentation products of dietary polysaccharides (Fig. 5) (60). Similarly, the genome sequence of Bifidobacterium longum revealed insights into the interaction of bifidobacteria with their host, as genes encoding polypeptides with homology to glycoprotein-binding fimbriae are present in the genome. Moreover, a eukaryotic-type serine protease inhibitor is encoded in the genome and could be involved in the reported immunomodulatory activity of bifidobacteria (61).

Recently, the complete genomes of L. plantarum (3.3 Mbp) and L. johnsonii (2.0 Mbp) were compared, revealing that these genomes have only 28 regions with conservation of gene order, encompassing approximately 0.75 Mbp (65). Notably, these regions are not co-linear, indicating major chromosomal rearrangements. Moreover, metabolic reconstruction indicated many differences between these two lactobacilli, as numerous enzymes involved in sugar metabolism and the biosynthesis of amino acids, nucleotides, fatty acids and cofactors are lacking in L. johnsonii. Interestingly, major differences were also seen in the number and types of putative extracellular proteins, which could play a role in host-microbe interactions in the GI tract. The differences between L. plantarum and L. johnsonii, both in genome organization and gene content, are exceptionally large for two bacteria of the same genus, emphasizing the complexity and diversity of the Lactobacillus genus (65).

Overall, the availability and comparison of bacterial genome sequences and their annotated functions provides valuable clues towards the survival strategy of these bacteria during their residence in the human GI tract. Additionally, these complete genome sequences are powerful tools for the convenient and effective interpretation of the data generated by the in vitro and in vivo screening procedures described above. Moreover, comparative genomics can provide important insight in diversity, evolutionary relationship and functional variation between bacteria, which might eventually generate a comprehensive view of the behavior of microbes during residence in the human GI-tract.

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