Bacillus subtilis can form various types of spatially organised communities on surfaces, such as colonies, pellicles and submerged biofilms. These communities share similarities and differences, and phenotypic heterogeneity has been reported for each type of community. Here, we studied spatial transcriptional heterogeneity across the three types of surface-associated communities. Using RNA-seq analysis of different regions or populations for each community type, we identified genes that are specifically expressed within each selected population. We constructed fluorescent transcriptional fusions for 17 of these genes, and observed their expression in submerged biofilms using time-lapse confocal laser scanning microscopy (CLSM). We found mosaic expression patterns for some genes; in particular, we observed spatially segregated cells displaying opposite regulation of carbon metabolism genes (gapA and gapB), indicative of distinct glycolytic or gluconeogenic regimes coexisting in the same biofilm region. Overall, our study provides a direct comparison of spatial transcriptional heterogeneity, at different scales, for the three main models of B. subtilis surface-associated communities.

Surface-associated multicellular assemblage is an important bacterial trait to withstand harsh environmental conditions. Bacillus subtilis is one of the most studied Gram-positive bacteria, serving as a model for the study of genetic pathways involved in the different steps of 3D biofilm formation. B. subtilis biofilm studies have mainly focused on pellicle formation at the air-liquid interface or complex macrocolonies formed on nutritive agar. However, only few studies focus on the genetic features of B. subtilis submerged biofilm formation and their link with other multicellular models at the air interface. NDmed, an undomesticated B. subtilis strain isolated from a hospital, has demonstrated the ability to produce highly structured immersed biofilms when compared to strains classically used for studying B. subtilis biofilms. In this contribution, we have conducted a multi-culturing comparison (between macrocolony, swarming, pellicle, and submerged biofilm) of B. subtilis multicellular communities using the NDmed strain and mutated derivatives for genes shown to be required for motility and biofilm formation in pellicle and macrocolony models. For the 15 mutated NDmed strains studied, all showed an altered phenotype for at least one of the different culture laboratory assays. Mutation of genes involved in matrix production (i.e., tasA, epsA-O, cap, ypqP) caused a negative impact on all biofilm phenotypes but favored swarming motility on semi-solid surfaces. Mutation of bslA, a gene coding for an amphiphilic protein, affected the stability of the pellicle at the air-liquid interface with no impact on the submerged biofilm model. Moreover, mutation of lytF, an autolysin gene required for cell separation, had a greater effect on the submerged biofilm model than that formed at aerial level, opposite to the observation for lytABC mutant. In addition, B. subtilis NDmed with sinR mutation formed wrinkled macrocolony, less than that formed by the wild type, but was unable to form neither thick pellicle nor structured submerged biofilm. The results are discussed in terms of the relevancy to determine whether genes involved in colony and pellicle formation also govern submerged biofilm formation, by regarding the specificities in each model.

Background The new SARS-CoV-2 variant VUI (202012/01), identified recently in the United Kingdom (UK), exhibits a higher transmissibility rate compared to other variants, and a reproductive number 0.4 higher. In the UK, scientists were able to identify the increase of this new variant through the rise of false negative results for the spike (S) target using a three-target RT-PCR assay (TaqPath kit). Methods To control and study the current coronavirus pandemic, it is important to develop a rapid and low-cost molecular test to identify the aforementioned variant. In this work, we designed primer sets specific to SARS-CoV-2 variant VUI (202012/01) to be used by SYBR Green-based RT-PCR. These primers were specifically designed to confirm the deletion mutations Δ69/Δ70 in the spike and the Δ106/Δ107/Δ108 in the NSP6 gene. We studied 20 samples from positive patients, 16 samples displayed an S-negative profile (negative for S target and positive for N and ORF1ab targets) and four samples with S, N and ORF1ab positive profile. Results Our results emphasized that all S-negative samples harbored the mutations Δ69/Δ70 and Δ106/Δ107/Δ108. This protocol could be used as a second test to confirm the diagnosis in patients who were already positive to COVID-19 but showed false negative results for S-gene. Conclusions This technique may allow to identify patients carrying the VUI (202012/01) variant or a closely related variant, in case of shortage in sequencing.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an outbreak of a pandemic worldwide. The spike (S) protein of SARS-CoV-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2 (ACE2), while the S2 subunit mediates viral cell membrane fusion with the cell membrane and subsequent entry into cells. Mutations in the spike protein (S) are of particular interest due to their potential for reduced susceptibility to neutralizing antibodies or increasing the viral transmissibility and infectivity. Recently, many mutations in the spike protein released new variants, including the Delta and Kappa ones (known as the Indian variants). The variants Delta and Kappa are now of most recent concern because of their well-increased infectivity, both a spin-off of the B.1.617 lineage, which was first identified in India in October 2020. This study employed homology modeling to probe the potential structural effects of the mutations. It was found that the mutations, Leu452Arg, Thr478Lys, and Glu484Gln in the spike protein increase the affinity for the hACE2 receptor, which explains the greater infectivity of the SARS-Cov-2 B.1.617 (Indian Variant).

Bacillus subtilis has been extensively used to study the molecular mechanisms behind the development and dispersal of surface bacterial multicellular communities. Well-structured spatially organised communities (colony, pellicle, and submerged biofilm) share some similarities, but also display considerable differences at the structural, chemical and biological levels. To unveil the spatial transcriptional heterogeneity between the different communities, we analysed by RNA-seq nine spatio-physiological populations selected from planktonic and spatially organised communities. This led to a global landscape characterisation of gene expression profiles uncovering genes specifically expressed in each compartmental population. From this mesoscale analysis and using fluorescent transcriptional reporter fusions, 17 genes were selected and their patterns of expression reported at single cell scale with time-lapse confocal laser scanning microscopy (CLSM). Derived kymographs allowed to emphasise spectacular mosaic gene expression patterns within a biofilm. A special emphasis on oppositely regulated carbon metabolism genes ( gapA and gapB ) permitted to pinpoint the coexistence of spatially segregated bacteria under either glycolytic or gluconeogenic regime in a same biofilm population. Altogether, this study gives novel insights on the development and dispersal of B. subtilis surface-associated communities.