Maximum depth sequencing reveals an ON/OFF replication slippage switch and apparent in vivo selection for bifidobacterial pilus expression

Abstract

The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in “beneficial” gut commensals.

HMO-primed bifidobacteria exhibit enhanced ability to adhere to intestinal epithelial cells

“…Therefore, the identified pilin clusters in B. bifidum R0071 were searched for the presence of a G-tract up to 500 bp upstream and 200 bp downstream of the predicted gene start in the first gene of the operon. In comparison to PRL2010, which harbours only one G-tract containing pilus gene cluster (Penno et al, 2022), polyG sequences were found to be present in two pilus-specifying gene clusters in B. bifidum R0071. A G-tract was identified in the pil3 gene cluster (BBIF_0313-0315) at the position 363939-36394911 (11 base pairs) and in the pil1 gene cluster (BBIF_1805-1807) at the position 2216145 to 2216157 (13 base pairs).…”

“…We confirm that polyG sequences were found downstream of the predicted gene start in Pil1 and Pil3, but not Pil2. The identification of polyG sequences is noteworthy, as productive replication slippage at G-tract sequences may be an important mediator of sortasedependent pili expression and subsequent colonization of bifidobacterial strains within the gastrointestinal tract (Penno et al, 2022). Importantly, significant upregulation of the pilus proteins within the B. bifidum Pil1 and Pil3 clusters was observed following exposure of the bifidobacteria to HMO.…”

“…In B. R0071 we identified six putative sortasedependent pilus proteins arranged in three pilin clusters at BBIF_1805-1807 (pil1: BBPR_1820-1822 in PRL2010), BBIF_1693-1,695 (pil2: BBPR_1707-1709) and BBIF_0313-0315 (pil3: BBPR_0282-0284). A recent study has established that the presence of a stretch of polyG influences the on-off switch in the expression of these pili genes (Penno et al, 2022). Therefore, the identified pilin clusters in B. bifidum R0071 were searched for the presence of a G-tract up to 500 bp upstream and 200 bp downstream of the predicted gene start in the first gene of the operon.…”