The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore's nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spore's inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEa's amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.T he spoVA operon of Bacillus subtilis is expressed only in the developing spore during sporulation and encodes seven proteins, with the genes in the order spoVAA, -B, -C, -D, -Eb, -Ea, and -F. At least five of these SpoVA proteins, SpoVAA, -B, -C, -D, and -Eb, are necessary for normal B. subtilis spore formation (1, 2). These proteins are essential for the uptake of dipicolinic acid (DPA) into the dormant spore (3-7); in turn, DPA is essential for spore stability, as DPA-less B. subtilis spores germinate spontaneously after their release from the sporangium and during spore purification (8). At least some SpoVA proteins are also essential for DPA release in spore germination, and one SpoVA protein, SpoVAD, binds DPA specifically, with this binding essential for DPA uptake in sporulation (6). SpoVAD is a soluble protein and has no obvious signal peptide or membrane-spanning or anchor sequences. However, assessment of SpoVAD's location and accessibility in spores indicates that this protein is on the outer surface of the spore's inner membrane (IM) (4, 9, 10).In contrast to SpoVAD, SpoVAA, -B, -C, -Eb, and -F appear to be integral membrane proteins on the basis of predictions from primary sequences and in some cases the localization of proteins expressed in growing bacteria (3,11,12). Although SpoVAF exhibits significant sequence identity to the A subunits of the spore's nutrient germinant receptors (GRs) (2), SpoVAA, -B, -C, -Eb, and -Ea exhibit no significant identity to known proteins. However, SpoVAEa is also predicted to be a soluble protein. Consequently, in this work, we have examined the location an...

We determined the presence of class 1 integrons related to the acquisition of resistance to antimicrobials in Aeromonas spp. isolated from individuals with diarrhea. Species were identified as A. caviae, A. hydrophila, A. veronii and A. media using PCR-RFLP of the 16S rDNA. Selected isolates were further characterized by ERIC-PCR. Resistance to chloramphenicol, aztreonam, tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid and streptomycin, among others, was determined using the Kirby-Bauer method. Integrons were detected by PCR amplification of the 5' conserved, variable, and 3' conserved regions. Sequencing of the variable regions revealed class 1 integrons with cassettes encoding resistance to trimethoprim (dfrA12, dfrA15, dfrB4), streptomycin/spectinomycin (aadA2, aadA1), oxacillin (oxa2) and chloramphenicol (catB3, cmlA4). Others had an open reading frame (orfD) or no insert at all. To our knowledge, this is the first description of the occurrence of genes cmlA4 and dfrA15 in Aeromonas class 1 integrons. Not all the integron-linked cassettes conferred their associated resistances, which suggests the inactivity of some cassettes. Most integrons were chromosomally located. The presence of class 1 integrons similar to those found in a wide variety of bacterial genera from different origins, including environmental and fish-borne Aeromonas, confirms the stability and horizontal transfer of these genetic elements.

Germination of Bacillus spores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore's inner membrane. GRs typically consist of three subunits referred to as A, B, and C, although functions of individual subunits are not known. Here we present the crystal structure of the N-terminal domain (NTD) of the A subunit of the Bacillus megaterium GerK 3 GR, revealing two distinct globular subdomains bisected by a cleft, a fold with strong homology to substratebinding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, and mutagenesis coupled with spore germination analyses support a proposed model that the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in spore germination. Our findings provide a conceptual framework for understanding the germinant recruitment mechanism by which GRs trigger spore germination.Bacillus | spores | spore germination | spore germinant receptor

Staphylococcus aureus is a commensal bacterium in humans and animals able to adapt to multiple environments. The aim of this study was to compare the genetic diversity and virulence profiles of strains of S. aureus isolated from food (29 strains), humans (43 strains), and animals (8 strains). 80 lipase-producing strains belonging to a biobank of 360 isolates, identified phenotypically as S. aureus, were selected. Confirmation of the species was made by amplifying the spA gene and 80% (64/80) of the strains were confirmed within this species. The virulence profile of each of the isolates was determined by PCR. The seA gene coding for enterotoxin A was found in 53.1% of the strains, the saK gene, which codes for Staphylokinase, was amplified in 57.8% of the strains, and, finally, the hlB gene coding for β-Hemolysin was amplified in 17.2%. The profile of antimicrobial resistance was determined by the Kirby Bauer method showing that the strains from food presented greater resistance to erythromycin (40.7%) and ciprofloxacin (18.5%) while in strains isolated from humans were to erythromycin (48.4%) and clindamycin (21.2%). Also, in strains from animals, a high resistance to erythromycin was observed (75%). The frequency of MRSA was 12.5% due to the presence of the mec gene and resistance to cefoxitin. Of the total strains, 68.7% were typed by PCR-RFLP of the coa gene using the AluI enzyme; derived from this restriction, 17 profiles were generated. Profile 4 (490 bp, 300 bp) was the most frequent, containing a higher number of strains with a higher number of virulence factors and antimicrobial resistance, which is associated with greater adaptation to different environments. In this study, a wide genetic diversity of strains of S. aureus from different foods, humans, and animals was found. This demonstrates evolution, genetic versatility, and, therefore, the adaptation of this microorganism in different environments.

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