Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR-LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.

Staphylococcus aureus capsule synthesis requires the precursor N-acetyl-glucosamine; however, capsule is synthesized during post-exponential growth when the availability of N-acetyl-glucosamine is limited. Capsule biosynthesis also requires aerobic respiration, leading us to hypothesize that capsule synthesis requires tricarboxylic acid cycle intermediates. Consistent with this hypothesis, S. aureus tricarboxylic acid cycle mutants fail to make capsule.Staphylococcus aureus produces two major exopolysaccharides: poly-N-acetylglucosamine (PNAG) and capsule. PNAG is synthesized by enzymes encoded by genes within the intercellular adhesin (ica) operon (icaADBC) (5, 15) during the exponential growth phase when C 6 carbohydrates are abundant (5,10,12). In contrast, capsular polysaccharides are predominantly synthesized during the post-exponential growth phase when C 6 carbohydrates are in short supply (13). The most commonly encountered S. aureus capsule types, 5 and 8, are synthesized from the amino sugars . Interestingly, the biosynthetic precursor of the capsular sugars is UDP-N-acetylglucosamine (11), the same amino sugar used in synthesizing PNAG. N-acetylglucosamine is synthesized from the glycolytic intermediate fructose 6-phosphate, and in a rich medium containing glucose, abundant levels of fructose 6-phosphate will be generated by glycolysis. As stated, capsule is most abundantly synthesized in the post-exponential phase of growth when glucose is growth limiting (4, 13).In the absence of glucose, fructose 6-phosphate can be synthesized by gluconeogenesis from the tricarboxylic acid (TCA) cycle intermediate oxaloacetate. To do this, oxaloacetate undergoes an ATP-dependent decarboxylation and phosphorylation by phosphoenolpyruvate carboxykinase (pckA) to generate phosphoenolpyruvate (PEP) (19). Gluconeogenesis can then generate fructose 6-phosphate from PEP, which can be used for UDP-activated N-acetylglucosamine biosynthesis. Support for the idea that post-exponential-phase capsule biosynthesis requires TCA cycle activity and PEP carboxykinase can be found in the observation that capsule is made during aerobic growth (6). In the Dassy and Fournier study, respiratory chain inhibitors were used to show that respiratory activity or a high proton motive force is required for post-exponentialgrowth-phase capsule biosynthesis (6). Although this was an excellent study, it was unclear as to why inhibiting respiratory activity prevented capsule biosynthesis. One consequence of inhibiting respiratory activity is the accumulation of reducing potential in the form of NADH. As NADH accumulates, the intracellular concentration of NAD ϩ decreases and the activity of NAD ϩ -requiring enzymes similarly decreases. During the post-exponential growth phase, the primary consumer of NAD ϩ is the TCA cycle; therefore, inhibiting respiratory activity inhibits TCA cycle activity. Further support that postexponential capsule biosynthesis requires TCA cycle activity can be seen in transposon mutagenesis studies that found inactivation...

5,6-Dimethylbenzimidazolyl-(DMB)-α-ribotide, [α-ribazole-5′-phosphate (α-RP)] is an intermediate in the biosynthesis of adenosylcobalamin (AdoCbl) in many prokaryotes. In such microbes, α-RP is synthesized by nicotinate mononucleotide (NaMN):DMB phosphoribosyltransferases (CobT in S. enterica), in a reaction that is considered to be the canonical step for the activation of the base of the nucleotide present in adenosylcobamides. Some Firmicutes lack CobT-type enzymes, but have a two-protein system comprised of a transporter (i.e., CblT) and a kinase (i.e., CblS) that can salvage exogenous α-ribazole (α-R) from the environment using CblT to take up α-R, followed by α-R phosphorylation by CblS. We report that Geobacillus kaustophilus CblT and CblS proteins restore α-RP synthesis in S. enterica lacking the CobT enzyme. We also show that a S. enterica cobT strain that synthesizes GkCblS ectopically makes only AdoCbl, even under growth conditions where the synthesis of pseudoCbl is favored. Our results indicate that S. enterica synthesizes α-R, a metabolite that had not been detected in this bacterium, and that GkCblS has a strong preference for DMB-ribose over adenine-ribose as substrate. We propose that in some Firmicutes DMB is activated to α-RP via α-R using an as-yet-unknown route to convert DMB to α-R, and CblS to convert α-R to α-RP.

Metal‐containing cyclic tetrapyrroles are widely distributed in nature, and together comprise the family of compounds frequently referred to as “the pigments of life,” namely cobamides (Cba, contain cobalt), chlorophylls (contain magnesium), hemes (contain iron), and factor F 430 (contains nickel). In comparison to these other modified tetrapyrroles, the B 12 architecture is slightly different. The chemistry of the cobalt ion is key to the function of the molecule as either a coenzyme or cofactor. Other differences include a contraction of the tetrapyrrole mainframe, which allows for tighter binding of the metal, and the presence of a lower nucleotide loop that provides an extra ligand for the metal. The biosynthesis of adenosylcobalamin requires the concerted effort of around 30 enzyme‐mediated steps tethered with the cellular provision of a range of cofactors and cobalt. The biosynthesis can be divided into two major sections, the first being the synthesis of the tetrapyrrole‐derived corrin ring and the second involving the synthesis and attachment of the lower nucleotide loop. In the last decade, much has been learned about how the vitamin form is converted to its biologically active coenzymic form, providing mechanistic answers into the events that overcome steep thermodynamic barriers in the formation of the covalent, organometallic bond between the cobalt ion of the ring and the 5′‐deoxyadenosyl upper ligand. Exciting recent discoveries regarding the biosynthesis of the lower ligand base 5,6‐dimethylbenzimidazole in aerobes and anaerobes have filled long‐standing gaps of knowledge, providing a solid platform for the analysis of what is likely to be a membrane‐anchored multienzyme complex.

Cobamides (Cbas) are coenzymes used by cells from all domains of life, but made de novo only by some bacteria and archaea. The last steps of the cobamide biosynthetic pathway activate the corrin ring and the lower ligand base, condense the activated intermediates, and dephosphorylate the product prior to the release of the biologically active coenzyme. In bacteria, a phosphoribosyltransferase (PRTase) enyzme activates the base into its α-mononucleotide. The enzyme from Salmonella enterica (SeCobT) has been extensively biochemically and structurally characterized. The crystal structure of the putative PRTase from the archaeum Methanocaldococcus jannaschii (MjCobT) is known but its function has not been validated. Here we report the in vivo and in vitro characterization of MjCobT. In vivo, in vitro, and phylogenetic data reported here show that MjCobT belongs to a new class of NaMN-dependent PRTase. We also show that the Synechococcus sp. WH7803 CobT protein has PRTase activity in vivo. Lastly, results of isothermal titration calorimetry and analytical ultracentrifugation analysis show that the biologically active form of MjCobT is a dimer, not a trimer, as suggested by its crystal structure.