Jamila Anba

Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages

The complete 31754 bp genome of bIL170, a virulent bacteriophage of Lactococcus lactis belonging to the 936 group, was analysed. Sixty-four ORFs were predicted and the function of 16 of them was assigned by significant homology to proteins in databases. Three putative homing endonucleases of the HNH family were found in the early region. An HNH endonuclease with zinc-binding motif was identified in the late cluster, potentially being part of the same functional module as terminase. Three putative structural proteins were analysed in detail and show interesting features among dairy phages. Notably, gpl12 (putative fibre) and gpl20 (putative baseplate protein) of bIL170 are related by at least one of their domains to a number of multi-domain proteins encoded by lactococcal or streptococcal phages. A 110- to 150-aa-long hypervariable domain flanked by two conserved motifs of about 20 aa was identified. The analysis presented here supports the participation of some of these proteins in host-range determination and suggests that specific adsorption to the host may involve a complex multi-component system. Divergences in the genome of phages of the 936 group, that may have important biological properties, were noted. Insertions/deletions of units of one or two ORFs were the main source of divergence in the early clusters of the two entirely sequenced phages, bIL170 and sk1. An exchange of fragments probably affected the regions containing the putative origin of replication. It led to the absence in bIL170 of the direct repeats recognized in sk1 and to the presence of different ORFs in the ori region. Shuffling of protein domains affected the endolysin (putative cell-wall binding part), as well as gpl12 and gpl20.

Streptococcus thermophilus Is Able To Produce a β-Galactosidase Active during Its Transit in the Digestive Tract of Germ-Free Mice

ABSTRACT This work presents data on the application of a bacterial luciferase used to monitor gene expression of Streptococcus thermophilus in the digestive tract. The main result is that the bacterium was able to produce an active β-galactosidase in the digestive tract, although it did not multiply during its transit. This production was enhanced when lactose (the inducer) was added to the diet.

Differential Activities of Four Lactobacillus casei Promoters during Bacterial Transit through the Gastrointestinal Tracts of Human-Microbiota-Associated Mice

ABSTRACT In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novo-synthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.

The Peptidyl-Prolyl Isomerase Motif Is Lacking in PmpA, the PrsA-Like Protein Involved in the Secretion Machinery of Lactococcus lactis

ABSTRACT The prsA-like gene from Lactococcus lactis encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in Bacillus subtilis, was characterized. This gene, annotated pmpA, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in L. lactis removed the degradation products previously observed with the Staphylococcus hyicus lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for B. subtilis, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of L. lactis in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of B. subtilis and PrtM of L. lactis, the L. lactis PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl cis/trans-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in Streptococcaceae, the family to which L. lactis belongs. PLP from Streptococcus pneumoniae and Enterococcus faecalis possess this signature, but those of L. lactis, Streptococcus pyogenes, and Streptococcus mutans do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in L. lactis and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.

Β-galactosidase production by Streptococcus thermophilus is higher in the small intestine than in the caecum of human-microbiota-associated mice after lactose supplementation

Transit kinetics and survival rates of a bacterial species from yoghurt (i.e. Streptococcus thermophilus strain FBI3) were examined in different digestive compartments of gnotoxenic and human-microbiota-associated mice. The production of the lactose-hydrolysing enzyme (i.e. beta-galactosidase) was also investigated within the digestive tract, using a chromosomal reporter system based on luciferase genes from Photorhabdus luminescens under the control of the plac promoter. In both mice models, S. thermophilus cells transited within 2 h from the stomach to the caecum-colon compartment of the digestive tract where they displayed a survival rate of nearly 100 %. In gnotoxenic mice, luciferase activity was found to increase in the second half of the small intestine and in the caecum-colon compartment when lactose was added to the drinking water provided to the animals. In human-microbiota-associated mice drinking lactose, luciferase activity was similarly increased in the second half of the small intestine but was drastically reduced in the caecum-colon compartment. This feature could be ascribed to the presence of the resident human microbiota.

Metabolic Adaptation of Lactococcus lactis in the Digestive Tract: The Example of Response to Lactose

Lactococcus lactis is a model of food-grade lactic acid bacterium, which can durably colonize the digestive tract of germ-free mice. To study in vivo the bacterial adaptation to a novel nutritional resource brought by alimentation, the lactose-catabolizing strain IL2661 of L. lactis was established in monoxeny in mice. Half of the mice then received a lactose-rich diet. The mouse has no efficient intestinal lactase and is well adapted to a follow-up of the metabolic activity of microbial origin. The analysis of lactose and lactate in the feces suggested that L. lactis was able to use lactose in vivo. We developed a proteomic approach to evaluate in deeper details the metabolic response of the bacterium. We observed that L. lactis switched its metabolism to use the novel carbon source and reduced the level of proteins involved in an alternative mode of ATP production. In parallel, we also found that the amount of proteins involved in transcriptional regulation, transport and catabolism decreased in the presence of lactose. The proteome analysis informed us about the resources used by the bacteria in absence of lactose. In competition experiments, we found that the metabolic adaptation gives a strong ecological advantage to the bacteria able to efficiently utilize lactose.