Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus: a chronicle of evolution in action

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation. RESULTS: Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp.. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre. CONCLUSIONS: The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor

The bacterial Mfd protein prevents DNA damage induced by the host nitrogen immune response in a NER-independent but RecBC-dependent pathway

Production of reactive nitrogen species is an important component of the host immune defence against bacteria. Here, we show that the bacterial protein Mfd (Mutation frequency decline), a highly conserved and ubiquitous bacterial protein involved in DNA repair, confers bacterial resistance to the eukaryotic nitrogen response produced by macrophage cells and during mice infection. In addition, we show that RecBC is also necessary to survive this stress. The inactivation of recBC and mfd genes is epistatic showing that Mfd follows the RecBC repair pathway to protect the bacteria against the genotoxic effect of nitrite. Surprisingly given the role of Mfd in transcription-coupled repair, UvrA is not necessary to survive the nitrite response. Taken together, our data reveal that during the eukaryotic nitrogen response, Mfd is required to maintain bacterial genome integrity in a NER-independent but RecBC-dependent pathway

Frequency of deletion formation decreases exponentially with distance between short direct repeats

International audienc

<em>csp</em>-like genes of <em>Lactobacillus delbrueckii</em> ssp. <em>bulgaricus</em> and their response to cold shock

International audienceThe two csp-like genes from the lactic acid bacterium Lactobacillus delbrueckii ssp. bulgaricus were characterized and designated cspA and cspB. The gene cspA has been identified using a polymerase chain reaction (PCR)-based approach with degenerated primers and further characterized using an inverse PCR strategy. cspA encodes a protein of 65 amino acid residues which displays between 81 and 77% identity with proteins CspL and CspP of Lactobacillus plantarum. cspB has been identified as a cspA ortholog using the partial sequence of the L. bulgaricus ATCC11842. cspB encodes a protein of 69 amino acids which has 42% identity with CspA. Northern blot analyses showed that cspA is transcribed as a single gene and that its transcription increased after a temperature downshift from 42 to 25 degrees C. In contrast, cspB is part of an operon transcribed at constant level irrespective of the temperature. These results indicate that cspA encodes the only Csp-like protein of L. bulgaricus induced by a downshift of temperature

The cytotoxic potential of Bacillus cereus strains of various origins

https://doi.org/10.1016/j.fm.2021.103759 B. cereus is a human pathogen associated with food poisoning leading to gastrointestinal disorders, as well as local and severe systemic infections. The pathogenic spectrum of B. cereus ranges from strains used as probiotics in humans to lethal highly toxic strains. In this study, we gathered a collection of 100 strains representative of the pathological diversity of B. cereus in humans, and characterized these strains for their cytotoxic potential towards human cells. We analyzed the correlation between cytotoxicity to epithelial and macrophage cells and the combination of 10 genes suspected to play a role during B. cereus virulence. We highlight genetic differences among isolates and studied correlations between genetic signature, cytotoxicity and strain pathological status. We hope that our findings will improve our understanding of the pathogenicity of B. cereus, thereby making it possible to improve both clinical diagnosis and food safety

NO IC 50 on <i>B</i>. <i>cereus</i> mutants.

<p>NO IC 50 on <i>B</i>. <i>cereus</i> mutants.</p

In the context of NO stress, we show that <i>mfd</i> and <i>recBC</i>/<i>addAB</i> deletions are epistatic.

<p>As RecBC participates in the repair of Double Strand Breaks (DSB), this indicates that Mfd also participates in DSB repair. It is unlikely that Mfd prevents DSBs because, in that case, RecBC would be essential for DNA repair in a <i>mfd</i> mutant. By contrast, we hypothesized that following the DSBs induced by NO exposure, RecBC has not directly access to Double strand ends (DSEs). Thus, Mfd would act first by removing the RNAP blocked on DNA lesions. Then the RecBCD complex can be recruited to repairs the DSB. RecBCD unwinds the DNA helix and degrades single strands. When RecBDC encounters a Chi site on the DNA, the degradation of the 5’ terminus is enhanced leaving a 3’ overhang and leads to the formation of an ssDNA. RecA binds to ssDNA and promotes repair by recombination with a homologous molecule of DNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163321#pone.0163321.ref050" target="_blank">50</a>].</p

Role of Mfd in <i>in vivo</i> survival following NO stress.

<p>C57/Bl6/Sev 129 mice (Wt Mice) and NOS2-/- mice (iNOS-KO mice) were inoculated intranasally with <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant bacteria (5.10⁶/mice). Wild type and <i>Δmfd</i> mutant strains were recovered from the lung (A) and from the brain (B) following mice death. The cfu recovered post infection was calculated by plating the bacteria on LB agar plates. For each mouse, the same symbol is used for lung (A) and brain (B) values. Cfu is shown for 5 wild type mice infected with Bc407, 4 wild type mice infected with Δ<i>mfd</i>, 3 KO mice infected with Bc407 and 3 KO mice infected with Δ<i>mfd</i>.</p

Phenotypical analysis of the Δ<i>mfd</i> mutant in mutagenic conditions.

<p>(A) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were inoculated in LB medium at a starting optical density (OD) of 0.07 and grown at 25°C with agitation. The OD was measured every hour at 600 nm. This graph represents representative growth curves out of at least five independent experiments. (B) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown at 37°C under agitation until mid exponential growth phase. Serial dilutions were plated on agar plates containing 50 ng/mL mitomycin C. Plates were incubated ON at 37°C and bacterial survival was assessed by observing the growth zone. Images correspond to a representative example out of at least 3 independent experiments done in duplicates. (C) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown at 37°C under agitation until mid exponential growth phase. Serial dilutions were plated on agar plates and exposed to UV light for 0 to 15 seconds at 5J/m². Plates were incubated ON at 37°C and bacterial survival was assessed by observing the growth zone. Images correspond to a representative example out of at least 3 independent experiments done in duplicates. (D) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown in LB medium until entry into stationary growth phase. Culture supernatant was filtered and added to HeLa cells. Cytotoxicity was measured by the trypan blue method after 2 h of incubation. Results are means of three independent experiments. (E) Bacterial strains were grown in LB medium at 37°C under agitation or without agitation (semi anaerobiosis). This graph represents representative growth curves out of at least three independent experiments. (F) Bacterial strains were grown in LB medium at 37°C under agitation, then diluted with the pH adjusted to 5, 6 or 7. Cfu were calculated after 24 h of growth by plating serial dilutions on LB agar plates. Results are means of at least three independent experiments. (G) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were cultured in LB medium for 24 h in the presence of the anti microbial peptide cecropin A. Cfu were calculated by plating serial dilutions on LB agar plates. Results are means of three independent experiments.</p