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A novel α1-2-fucosyltransferase from Thermosynechococcus elongatus BP-1 (Te2FT) with high fucosyltransferase activity and low donor hydrolysis activity was discovered and characterized. It was used in an efficient one-pot multienzyme (OPME) fucosylation system for the high-yield synthesis of human blood group H antigens containing β1-3-linked galactosides and an important human milk oligosaccharide (HMOS) lacto-N-fucopentaose I (LNFP I) on preparative and gram scales. LNFP I was shown to be selectively consumed by Bifidobacterium longum subsp. infantis but not Bifidobacterium animalis subsp. lactis and is a potential prebiotic.

Human milk is a unique and complex fluid that provides infant nutrition and delivers an array of bioactive molecules that serve various functions. Glycans, abundant in milk, can be found as free oligosaccharides or as glycoconjugates. Milk glycans are increasingly linked to beneficial outcomes in neonates through protection from pathogens and modulation of the immune system. Indeed, these glycans influence the development of the infant and the infant-gut microbiota. Bifidobacterium species commonly are enriched in breastfed infants and are among a limited group of bacteria that readily consume human milk oligosaccharides (HMOs) and milk glycoconjugates. Given the importance of bifidobacteria in infant health, numerous studies have examined the molecular mechanisms they employ to consume HMOs and milk glycans, thus providing insight into this unique enrichment and shedding light on a range of translational opportunities to benefit at-risk infants.

Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC–MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host–microbe associations and membrane protein activities.

Mammalian milk is a source of antimicrobial compounds such as xanthine oxidase (XO). The interplay of infant saliva, which contains the substrates for XO activity, and human milk containing XO has been recently shown to inhibit the growth of pathogenic bacteria. Based on the complex and protective mechanism observed in human milk, we hypothesized that bovine milk XO operates similarly, thus representing an opportunity to investigate its functionality in broader health implications. We demonstrated that bovine milk-hypoxanthine mixture (0 to 400 μM) inhibited several Gram-negative and -positive bacterial pathogens in a dose-dependent manner. Kinetic experiments revealed that XO catalyzed hypoxanthine reduction (K(m), 58.0 μM; V(max), 5.1 μmol(−1) min(−1) mg) resulted in the production of antimicrobial hydrogen peroxide. These results demonstrate that the antimicrobial properties of bovine milk XO are similar to those of human milk XO with significant implications for the development of novel products targeting infant health.

Consumption of mothers’ milk is associated with reduced incidence and severity of enteric infections, leading to reduced morbidity in breastfed infants. Fucosylated and sialylated human milk oligosaccharides (HMO) are important for both direct antimicrobial action — likely via a decoy effect — and indirect antimicrobial action through commensal growth enhancement. Bovine milk oligosaccharides (BMO) are a potential source of HMO-mimics as BMO resemble HMO; however, they have simpler and less fucosylated structures. BMO isolated at large scales from bovine whey permeate were modified by the addition of fucose and/or sialic acid to generate HMO-like glycans using high-yield and cost-effective one-pot multienzyme approaches. Quadrupole time-of-flight LC/MS analysis revealed that 22 oligosaccharides were synthesized and 9 had identical composition to known HMO. Preliminary anti-adherence activity assays indicated that fucosylated BMO decreased the uptake of enterohemorrhagic Escherichia coli O157:H7 by human intestinal epithelial Caco-2 cells more effectively than native BMO.

Bacteria can exist in metabolically inactive states that allow them to survive conditions that are not conducive for growth. Such dormant cells may sense when conditions have improved and re-initiate growth, lest they be outcompeted by their neighbours. Growing bacteria turn over and release large quantities of their cell walls into the environment. Drawing from recent work on the germination of Bacillus subtilis spores, we propose that many microorganisms exit dormancy in response to cell wall muropeptides.

According to the prerecruitment hypothesis, Escherichia coli SoxS activates the transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase (RNAP) that scan the chromosome for class I and class II SoxS-dependent promoters. We showed previously that the alpha subunit's C-terminal domain plays a role in activating both classes of promoter by making protein-protein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the prerecruitment hypothesis. Here, we identified seven single-alanine substitutions of the region 4 of sigma(70) (sigma(70) R4) of RNAP that reduce SoxS activation of class II promoters. With genetic epistasis tests between these sigma(70) R4 mutants and positive control mutants of SoxS, we identified 10 pairs of amino acids that interact with each other in E. coli. Using the yeast two-hybrid system and affinity immobilization assays, we showed that SoxS and sigma(70) R4 can interact in solution (i.e., "off-DNA"). The interaction requires amino acids of the class I/II (but not the class II) positive control surface of SoxS, and five amino acids of sigma(70) R4 that reduce activation in E. coli also reduce the SoxS-sigma(70) R4 interaction in yeast. One of the epistatic interactions that occur in E. coli also occurs in the yeast two-hybrid system (i.e., off-DNA). Importantly, we infer that the five epistatic interactions occurring in E. coli that require an amino acid of the class II surface occur "on-DNA" at class II promoters. Finding that SoxS contacts sigma(70) R4 both off-DNA and on-DNA is consistent with the prerecruitment hypothesis. Moreover, SoxS is now the first example of an E. coli transcriptional activator that uses a single positive control surface to make specific protein-protein contacts with two different subunits of RNAP.

Here, we report that the model Gram-positive organism, Bacillus subtilis, expresses and secretes a muralytic enzyme, YocH, in response to cell wall-derived muropeptides derived from growing cells but not lysed cells. This induction is dependent on PrkC, a membrane Ser/Thr kinase that binds to peptidoglycan and that belongs to a broadly conserved family including the essential PknB kinase of M. tuberculosis. YocH stimulates its own expression in a PrkC-dependent manner demonstrating the presence of an autoregulatory loop during growth. Cells lacking YocH display a survival defect in stationary phase but enzymes secreted by other cells in the culture rescue this defect. The essential translation factor EF-G is an in vivo substrate of PrkC and this phosphorylation occurs in response to muropeptides. Therefore, we hypothesize that YocH is used by the bacterium to digest peptidoglycan released by other bacteria in the milieu and that the presence of these fragments is detected by a membrane kinase that modifies a key regulator of translation as well as to stimulate its own expression.

Superdormant spores of Bacillus cereus and Bacillus subtilis germinated just as well as dormant spores with pressures of 150 or 500 MPa and with or without heat activation. Superdormant B. subtilis spores also germinated as well as dormant spores with peptidoglycan fragments or bryostatin, a Ser/Thr protein kinase activator.

Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses.

When Escherichia coli encounter redox-cycling compounds that endogenously generate superoxide, the cell's defense response is initiated by the de novo synthesis of SoxS, which then activates transcription of the genes of the SoxRS regulon. Recently, we showed that after the oxidative stress is relieved, the SoxRS system resets by an active process wherein SoxS synthesis ceases and the intrinsically unstable SoxS protein is rapidly degraded, primarily by Lon protease. Here, we use deletion mutants and a library of alanine-stretch mutants of the entire protein to identify the SoxS features responsible for Lon-dependent proteolysis in vivo. We found that the 17 amino acid residues at the SoxS N terminus play the primary role in protease recognition and that the addition of the N-terminal 21 residues of SoxS to the otherwise stable green fluorescent protein is sufficient to signal the chimera for Lon-dependent degradation. With a minimal in vitro degradation system, we confirm the intrinsic instability of SoxS and the sequence requirements for Lon-dependent degradation. Lastly, we demonstrate that the addition of a peptide comprised of the 21 N-terminal amino acid residues of SoxS is able to inhibit specifically the in vitro proteolysis of SoxS.

Escherichia coli SoxS, the direct transcription activator of the SoxRS (superoxide) regulon, is intrinsically unstable with an in vivo half-life of approximately 2 min. Overexpression of SoxS is lethal, but mutations interfering with DNA binding relieve the toxicity. Here, we determined the effects on the half-life of SoxS of alanine substitutions that confer defects in positive control, i.e. transcription activation, or in specific DNA binding. We found that both types of mutations render SoxS more unstable than the wild-type protein, as if 'soxbox' DNA and RNA polymerase serve as stabilizing ligands in vivo that protect SoxS from degradation by Lon, the protease shown previously to be primarily responsible for its turnover. Indeed, we found that the addition of soxbox DNA or RNA polymerase to an in vitro degradation system decreases the rate of SoxS proteolysis by Lon protease. To the best of our knowledge, these are the first examples of target DNA and RNA polymerase serving as ligands that inhibit the turnover of an unstable transcription activator.

Summary In Escherichia coli, the SoxRS regulon confers resistance to redox-cycling compounds, and the Mar regulon provides a defence against multiple antibiotics. The response regulators, SoxS and MarA, are synthesized de novo in response to their inducing signals and directly activate transcription of a common set of target genes. Although the mechanisms of transcription activation by SoxS and MarA have been well studied, little is known about how the systems are shut-off once the inducing stress has subsided, except that de novo synthesis of the regulators is known to cease almost immediately. Here, we induced the SoxRS regulon and determined that, upon removal of the inducer, expression of the regulon's genes quickly returns to the preinduced level. This rapid shut-off indicates that the system is reset by an active process. We found that SoxS is unstable and infer that SoxS degradation is responsible for the rapid return of the system to the ground state upon removal of the inducing signal. We also found that MarA is unstable and that the instability of both proteins is intrinsic and unregulated. We used null mutations of protease genes to identify the proteases involved in the degradation of SoxS and MarA. Among single protease mutations, only lon mutations increased the half-life of SoxS and MarA. In addition, SoxS appeared to be nearly completely stable in a lon ftsH double mutant. Using hexahistidine tags placed at the respective ends of the activators, we found that access to the amino-terminus is essential for the proteolytic degradation.

Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.

Certain Gram-positive bacteria—as exemplified by Bacillus anthracis, the causative agent of anthrax—can produce dormant and environmentally resistant spores under conditions of nutritional limitation. These spores exit from dormancy via the process of germination that is triggered by exposure to specific molecules.[1] While the precise chemical nature of these molecules, known as germinants, varies according to the organism, they are typically nutrients.[2] Recently, we reported that supernatants from cultures of growing bacteria and constituents of the cell wall could serve as germinants of dormant B. subtilis and B. anthracis spores.[3] Since fragments of the cell wall are released in the course of bacterial growth, the presence of these molecules in the milieu as germination signals might be physiologically relevant.

SoxS is the transcription activator of the SoxRS regulon. Despite being synthesized de novo in response to oxidative stress and despite the large disparity between the number of SoxS binding sites and the number of SoxS molecules per cell, SoxS-dependent promoters are rapidly activated after the onset of the stress. With the usual recruitment/post-recruitment mechanisms being unsuitable for activating gene expression under these conditions, we previously proposed that SoxS functions by "pre-recruitment". In pre-recruitment, SoxS forms SoxS-RNA polymerase binary complexes, which use the DNA binding properties of SoxS and sigma(70) to distinguish SoxS-dependent promoters from housekeeping promoters and from the large number of sequence-equivalent but functionally irrelevant SoxS binding sites. With previous work in Escherichia coli having indicated that the most likely target on RNA polymerase for interaction with SoxS is the C-terminal domain of alpha, we investigated the interaction directly with the yeast two-hybrid system. We found that SoxS interacts with the alphaCTD and that SoxS positive control mutations disrupt the interaction. Moreover, single alanine substitutions of the alphaCTD that reduce or enhance SoxS activation in E.coli reduce or enhance the interaction between SoxS and the alphaCTD in yeast. Significantly, the critical amino acid residues lie in and around the DNA binding determinant of the alphaCTD, the first example of an activator contacting this determinant. These interactions were confirmed with an affinity immobilization assay. Lastly, we found that SoxS induction interferes with utilization of the UP element of an rRNA promoter. Thus, by functioning as a co-sigma factor that interacts with the DNA binding determinant of the alphaCTD, SoxS diverts RNA polymerase from UP-containing promoters to SoxS-activatable promoters.

In Escherichia coli, the SoxRS regulon confers resistance to redox-cycling compounds, and the Mar regulon provides a defence against multiple antibiotics. The response regulators, SoxS and MarA, are synthesized de novo in response to their inducing signals and directly activate transcription of a common set of target genes. Although the mechanisms of transcription activation by SoxS and MarA have been well studied, little is known about how the systems are shut-off once the inducing stress has subsided, except that de novo synthesis of the regulators is known to cease almost immediately. Here, we induced the SoxRS regulon and determined that, upon removal of the inducer, expression of the regulon's genes quickly returns to the preinduced level. This rapid shut-off indicates that the system is reset by an active process. We found that SoxS is unstable and infer that SoxS degradation is responsible for the rapid return of the system to the ground state upon removal of the inducing signal. We also found that MarA is unstable and that the instability of both proteins is intrinsic and unregulated. We used null mutations of protease genes to identify the proteases involved in the degradation of SoxS and MarA. Among single protease mutations, only lon mutations increased the half-life of SoxS and MarA. In addition, SoxS appeared to be nearly completely stable in a lon ftsH double mutant. Using hexahistidine tags placed at the respective ends of the activators, we found that access to the amino-terminus is essential for the proteolytic degradation.

In response to the oxidative stress imposed by redox-cycling compounds like paraquat, Escherichia coli induces the synthesis of SoxS, which then activates the transcription of approximately 100 genes. The DNA binding site for SoxS-dependent transcription activation, the "soxbox," is highly degenerate, suggesting that the genome contains a large number of SoxS binding sites. To estimate the number of soxboxes in the cell, we searched the E. coli genome for SoxS binding sites using as query sequence the previously determined optimal SoxS binding sequence. We found approximately 12,500 sequences that match the optimal binding sequence under the conditions of our search; this agrees with our previous estimate, based on information theory, that a random sequence the size of the E. coli genome contains approximately 13,000 soxboxes. Thus, fast-growing cells with 4-6 genomes per cell have approximately 65,000 soxboxes. This large number of potential SoxS binding sites per cell raises the interesting question of how SoxS distinguishes between the functional soxboxes located within the promoters of target genes and the plethora of equivalent but nonfunctional binding sites scattered throughout the chromosome. To address this question, we treated cells with paraquat and used Western blot analysis to determine the kinetics of SoxS accumulation per cell; we also determined the kinetics of SoxS-activated gene expression. The abundance of SoxS reached a maximum of 2,500 molecules per cell 20 min after induction and gradually declined to approximately 500 molecules per cell over the next 1.5 h. Given that activation of target gene expression began almost immediately and given the large disparity between the number of SoxS molecules per cell, 2,500, and the number of SoxS binding sites per cell, 65,000, we infer that SoxS is not likely to activate transcription by the usual "recruitment" pathway, as this mechanism would require a number of SoxS molecules similar to the number of soxboxes. Instead, we propose that SoxS first interacts in solution with RNA polymerase and then the binary complex scans the chromosome for promoters that contain a soxbox properly positioned and oriented for transcription activation. We name this new pathway "pre-recruitment."

SummaryBacteria can respond to adverse environmental conditions by drastically reducing or even ceasing metabolic activity. They must then determine that conditions have improved before exiting dormancy, and one indication of such a change is the growth of other bacteria in the local environment. Growing bacteria release muropeptide fragments of the cell wall into the extracellular milieu, and we report here that these muropeptides are potent germinants of dormant Bacillus subtilis spores. The ability of a muropeptide to act as a germinant is determined by the identity of a single amino acid. A well-conserved, eukaryotic-like Ser/Thr membrane kinase containing an extracellular domain capable of binding peptidoglycan is necessary for this response, and a small molecule that stimulates related eukaryotic kinases is sufficient to induce germination. Another small molecule, staurosporine, that inhibits related eukaryotic kinases blocks muropeptide-dependent germination. Thus, in contrast to traditional antimicrobials that inhibit metabolically active cells, staurosporine acts by blocking germination of dormant spores.