By fermenting B. subtilis [1], are missing PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28192408 in A. flavithermus, STI-571 custom synthesis indicating that it cannot produce acetoin. In agreement with the experimental data [6], genome analysis indicates that A. flavithermus is able to utilize a variety of carbohydrates as sole carbon sources. It has at least four sugar phosphotransferase systems with predicted specificity for glucose, fructose, sucrose, and mannitol. Additionally, it encodes ABC-type transporters for ribose, glycerol-3-phosphate, and maltose, and several ABC-type sugar transporters of unknown specificity. A complete set of enzymes was identified for general carbohydrate metabolism (glycolysis, the TCA cycle, and the pentose phosphate pathway, but not the Entner-Doudoroff pathway). The A. flavithermus genome also contains a gene cluster (Aflv_2610-2618) that is very similar to the gene cluster associated with antibiotic production and secretion in many other Gram-positive bacteria [21], suggesting that A. flavithermus might be able to produce bactericidal peptides. It is not obvious which of these systems are relevant to the survival of A. flavithermus in silica solutions, but they might facilitate its growth in powdered milk and similar habitats.losses in the Geobacillus/Anoxybacillus branch include, among others, genes encoding the nitrogen regulatory protein PII, ABC-type proline/glycine betaine transport system, methionine synthase II (cobalamin-independent), sorbitolspecific phosphotransferase system, -xylosidase, and some dTDP-sugar metabolism genes (Table S3 in Additional data file 1). However, 62 gene gains were inferred as well, including several genes coding for cobalamin biosynthesis enzymes, methylmalonyl-CoA mutase, genes involved in assembly of type IV pili (Aflv_0630-0632), an uncharacterized ABC-type transport system, and 16 genes encoding uncharacterized conserved proteins (Table S3 in Additional data file 1). After the split of the Anoxybacillus and Geobacillus lineages, A. flavithermus continued to show strong genome reduction (-292 genes) compared to G. kaustophilus (-124 genes), losing, in particular, some genes of nitrogen and carbohydrate metabolism. In addition, A. flavithermus has apparently experienced less gene gain (+88) than G. kaustophilus (+158). The few genes likely acquired in the Anoxybacillus lineage include the clustered regularly interspaced short palindromic repeat (CRISPR)-associated genes (Aflv_0764-0771) that form an antisense RNA-based system of phage resistance, which is often associated with thermophily [23,24].Signal transductionBeing a free-living environmental microorganism, A. flavithermus encodes numerous proteins involved in signal transduction. These include 23 sensor histidine kinases and 24 response regulators (16 pairs of which are clustered in operons), 20 methyl-accepting chemotaxis proteins, 5 predicted eukaryotic-type Ser/Thr protein kinases, and 21 proteins involved in metabolism of cyclic diguanylate (cyclic (3′,5′)-dimeric guanosine monophosphate (c-di-GMP)), a recently recognized secondary messenger that regulates transition from motility to sessility and biofilm formation in a variety of bacteria [25]. Compared to other bacilli, this set is significantly enriched in chemotaxis transducers and c-diGMP-related proteins [26]. Anoxybacillus flavithermus encodes 12 proteins with the diguanylate cyclase (GGDEF) domain, 6 of which also contain the c-di-GMP phosphodiesterase (EAL) domain, and one combines GGDEF with an alternative c-di-GMP ph.