Contribution of Aggregation-Promoting Factor to Maintenance of Cell Shape in Lactobacillus gasseri 4B2

Abstract

Aggregation-promoting factor (APF) was originally described as a protein involved in the conjugation and autoaggregation of Lactobacillus gasseri 4B2, whose corresponding apf gene was cloned and sequenced. In this report, we identified and sequenced an additional apf gene located in the region upstream of the previously published one. Inactivation of both apf genes was unsuccessful, indicating that APF function may be essential for the cell. Overproduction of APF proteins caused drastic alteration in the cell shape of this strain. These cells were irregular, twisted, enlarged, and tightly bound in unbreakable clumps of chains. Down-regulation of APF synthesis was achieved by cloning of the apf2 promoter region on a high-copy-number plasmid, which recruited a putative apf activator. As a consequence, the shape of the corresponding recombinant cells was elongated (filamentous) and cell division sites were no longer visible. None of the induced changes in APF production levels was clearly correlated with modifications of the aggregation phenotype. This report shows, for the first time, that APF proteins are mainly critical for L. gasseri 4B2 cell shape maintenance.Lactic acid bacteria are normal inhabitants of the human oral cavity and gastrointestinal (GI) and urogenital tracts. Interest in the health-promoting effects of specific lactic acid bacterial strains has increased in recent years (10, 12). Survival and persistence of probiotic bacteria in the human GI tract is often reported to provide them a competitive advantage. One of the proposed mechanisms that could increase the potential of bacteria to survive and persist in the GI tract is their ability to aggregate (13). However, this hypothesis has never been confirmed.To study the importance of aggregation, we chose an aggregating strain, Lactobacillus gasseri 4B2 (previously classified as L. plantarum 4B2), whose 32-kDa aggregation-promoting factor (APF) has already been described (25). This protein was purified from the culture supernatant as one of the most abundant proteins. The function of APF as a factor of aggregation was shown in vitro. After three subsequent washing steps, L. gasseri 4B2 lost its ability to aggregate. Addition of purified APF or of filtered supernatant reaggregated the washed cells. Furthermore, the role of APF in conjugation was also demonstrated (25). The frequency of pAM␤1 conjugal transfer (among Lactobacillus species whose aggregation is APF dependent) was increased upon addition of L. gasseri 4B2 filtered supernatant to the mating mixture. Since neither the apf gene nor any apf mutant was available, the exact physiological role of APF could not be demonstrated. Subsequent N-terminal sequencing of the L. gasseri 4B2 APF protein enabled the cloning and sequencing of the apf gene (accession no. Y08498; L. Morelli et al., direct submission).Recently, we showed that four L. johnsonii and two L. gasseri strains contain two apf genes in their genomes (34). Analysis of the gene organization, amino acid composition, and physical propert...

Characterization of Lactobacillus coryniformis DSM 20001 T Surface Protein Cpf Mediating Coaggregation with and Aggregation among Pathogens

“…This indicated that E. coli produces the recombinant Cpf in a correctly folded conformation. Furthermore, all attempts to inactivate cpf and heterologously express cpf in L. reuteri or L. sakei failed (data not shown), indicating that Cpf may play an important role in cell surface composition, as recently demonstrated for Apf of L. gasseri (19).…”

“…The Apf proteins have recently been described as novel surface proteins (48). In L. gasseri the Apf is involved in the maintenance of the cell shape (19). On the other hand, little data are available about the coaggregation of lactobacilli and its mechanism.…”

In vitro characterization of aggregation and adhesion properties of viable and heat-killed forms of two probiotic Lactobacillus strains and interaction with foodborne zoonotic bacteria, especially Campylobacter jejuni

“…farciminis 3699 involves carbohydrate-lectin interactions and/or proteinaceous components present on the surface. Autoaggregation is a known property of some Lactobacillus isolates (Collado et al, 2007(Collado et al, , 2008 and allows the formation of biofilm in the gastrointestinal tract which contributes, as a physico-chemical barrier, to the preservation of the intestinal wall, along with the stimulation of the immune system (Jankovic et al, 2003;Castagliuolo et al, 2005;Voltan et al, 2007;Turpin et al, 2010), and to protection against colonization by pathogenic bacteria (Collado et al, 2007(Collado et al, , 2008. Furthermore, bacteria with a high autoaggregation potential will more easily colonize the intestinal and urogenital tracts (Boris et al, 1998;Cesena et al, 2001;Collado et al, 2005Collado et al, , 2007Collado et al, , 2008.…”

The predicted secretome of Lactobacillus plantarum WCFS1 sheds light on interactions with its environment

“…Four L. plantarum proteins contain a C-terminal domain of approximately 60 amino acids characterized by conserved tryptophan and tyrosine residues. Experimental studies in Lactobacillus gasseri and Lactobacillus johnsonii suggest that proteins containing this domain are involved in aggregation and/or maintenance of cell shape (Jankovic et al, 2003;Ventura et al, 2002).…”