E Although many studies have reported on the H2O2 production by species of the L. acidophilus group, our understanding of the enzymatic reactions and mechanisms underlying these observations remains limited to the notion that NADH and flavin are involved in the reaction (35, 36) and that it is catalyzed by a protein that is constitutively expressed (8). De Vos A subsequent double-blind, placebo-controlled, randomized, cross-over study protocol was used to determine the small intestinal transcriptional responses in human volunteers after consumption of a series of small-sized, bacteria-containing ‘sports drinks.’ These studies demonstrated that functionally coherent and distinct transcriptome responses are launched by the human intestinal mucosa following the consumption of different Lactobacillus species (L. acidophilus, L. casei and L. rhamnosus), which appear to be correlated with the previously reported effects of these lactobacilli on host physiology and health (P. van Baarlen, F. Troost, van der C. Meer, G. Hooiveld, M. Boekschoten, & M. Kleerebezem, unpublished data). Furthermore, two genome-wide genetic screens in L. reuteri (Walter et al., 2003) and L. plantarum (Bron et al., 2004a) exploiting in vivo expression technology revealed the induction of 3 and 72 genes in the mouse intestinal tract, respectively. (, Chaillou RJ De Keersmaecker (, Brooijmans (, Lee

In contrast to the apparent variability of WTA synthesis among lactobacilli, the conservation of at least one ltaS gene suggests that these species produce LTA consistently. DJ

Recently, the transcriptomes of L. plantarum WCFS1 in the cecum of mono-associated mice fed either a western-style (high fat, low fiber) or a standard chow diet (low fat, high fiber) were compared with in vitro transcriptomes (Marco et al., 2009). L Comstock Gordon MC S RJ

(, Billot-Klein (, O'Sullivan J Surprisingly, L. plantarum contains an racD orthologue, suggesting that aspartate racemase could be required for a metabolic function other than peptidoglycan biosynthesis in this species. The impact of a depletion of d-alanyl esters of TA was investigated in three of the four strains cited above by the inactivation of the dlt operon (Palumbo et al., 2006; Perea Velez et al., 2007; Walter et al., 2007); the dlt mutants had either strongly reduced or no d-alanyl esters, but the chain length was, respectively, threefold increased in a subpopulation of LTA of L. plantarum WCFS1, 1.7- and 8-fold reduced in the two subpopulations of LTA of L. rhamnosus GG and unaffected in L. reuteri 100-23. Moreover, multiple homologous proteins from one genome are found in some of these LaCOGs, such as the four different members of the cell-wall proteinase Prt family (LaCOG 90024) in L. casei, and the four paralogous genes for cell-surface hydrolases (LaCOG01138) in L. plantarum. (, Kleerebezem

SR Vaughan & M. Kleerebezem, unpublished data). Following the initial focus of bacterial genomics on pathogenic and paradigm laboratory species, the focus has shifted to encompass many industrially relevant and benign bacteria, including lactobacilli. (, Bron Kinoshita 3B). J CM S Gahan PW The genomic islands specific for strain GG encode approximately 80 proteins, including those involved in sugar metabolism and transport, and exopolysaccharide biosynthesis. S The natural habitat of lactobacilli ranges from dairy, meat and plant material fermentations to the oral cavity, and the genital and gastrointestinal tracts of humans and animals (Hammes & Vogel, 1995; Vaughan et al., 2002). The recent discovery of the LTA synthase LtaS [poly(Gro-P) polymerase] in S. aureus and B. subtilis (four paralogues) and their inactivation allowed specific blocking of LTA biosynthesis (Grundling & Schneewind, 2007; Schirner et al., 2009). LB Palva Construction of the L. johnsonii overexpression strains.The L. johnsonii expression plasmid pDP794 was constructed as follows: the predicted bidirectional terminator situated between the LJ_1125 and LJ_1126 genes of NCC 533 was amplified using primers O and P. This 359-bp amplicon was digested with the restriction enzymes HindIII and XhoI and cloned into similarly digested pNZ124 (40) to yield pNZ124-LJ_1125 trm. DB Wadstrom Slesarev

Hols (, Zoetendal TR Quivey H

Holo L T Chromatograms were analyzed using AZUR chromatography software (St. Martin D'Heres, France). B H

These results show that the LJ_0548-LJ_0549 operon encodes the observed NADH-dependent flavin reductase activity.

Van Der Meulen Perez-Martinez Buck The protein concentration is determined by absorption at 280 nm (black line). Altermann In addition, it addresses the nonproteinaceous building blocks of the Lactobacillus cell wall because they play a key role in interactions with the host.

(, Fozo However, this interesting impact of a variation in the LTA composition observed with the L. plantarum mutant seems species-specific, because the dlt mutant of L. rhamnosus is not affected compared with the wild type in similar in vitro immune modulation experiments (Perea Velez et al., 2007). Through autolysis in the host and cell-wall turnover, lactobacilli could release muramyl-peptides that are known to interact with receptors of the immune system. Mutation of these genes in L. johnsonii NCC 533 led to a strain that failed to produce H2O2 upon exposure to molecular oxygen. By contrast, the lactobacilli that are intrinsically resistant to vancomycin produce d-Lac in variable amounts as an end-product of fermentation (Table 2). Deletion of the predicted pox, lox, or nox genes did not significantly affect the level of hydrogen peroxide production after 1 h in these strains in comparison with the level produced by the parental strain NCC 533 (Fig. NOD1, NOD2, peptidoglycan recognition proteins (PGRPs), TLR2] of the host innate immune system (illustrated for NOD1 and NOD2 in Fig. SM

R

Brown AL MC Serata Here, H2O2 directly affected the activity of indoleamine 2,3-dioxygenase, which is an important immune modulator (32). Komatsuzawa The sortase (SrtA) enzyme is a transpeptidase that recognizes the LPxTG motif, cleaves the motif between the T and G residues and covalently attaches the threonine carboxyl group to the peptidoglycan (Marraffini et al., 2006). Hols (, Dobson RD Mannsfeld

V J K

During or after completion of the Sec-dependent translocation process, the C region becomes exposed to the extra-cytoplasmic side of the membrane. The active fraction obtained was digested with trypsin and analyzed using LC-MS/MS, using the fraction preceding this active fraction (and containing no activity) as a comparative negative control. J Van Dijl AJ Their addition to fermented foods is known to improve the storage stability, flavour, and texture of the products and, following food ingestion, they exert several beneficial ‘probiotic’ effects at the intestinal and immune-system levels. YS

C Hulst The observation that the deletion derivative NCC 9359 produces small amounts of H2O2 upon prolonged exposure to oxygen indicates that besides the NADH flavin reductase identified here, other H2O2-producing enzymes may exist in this species. K Cocconcelli J

WM O'Callaghan Molenaar