Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [32P]phosphoric acid to reveal a P3 intermediate. The 32P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis. 32P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein

Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus: sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-Gly3 substrates

Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH2-Gly3, sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH2-Gly3. In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate

Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium Actinomyces oris.

The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans-acting factors contributing to these processes. We report here a large-scale Tn5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis, and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn5 insertions in fimA, cafA, or srtC2, as expected; the latter were mapped to genes coding for uncharacterized proteins and various nuo genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of nuo genes and ubiE, a menaquinone C-methyltransferase-encoding gene downstream of the nuo locus, confirmed the pilus and coaggregation defects. Both nuoA and ubiE mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the ubiE mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the A. oris electron transport chain is biochemically linked to pilus assembly via oxidative protein folding.IMPORTANCE The Gram-positive actinobacterium A. oris expresses adhesive pili, or fimbriae, that are essential to biofilm formation and Actinomyces interactions with other bacteria, termed coaggregation. While the critical role of the conserved sortase machine in pilus assembly and the disulfide bond-forming catalyst MdbA in oxidative folding of pilins has been established, little is known about other trans-acting factors involved in these processes. Using a Tn5 transposon screen for mutants defective in coaggregation with Streptococcus oralis, we found that genetic disruption of the NADH dehydrogenase and menaquinone biosynthesis detrimentally alters pilus assembly. Further biochemical characterizations determined that menaquinone is important for reactivation of MdbA. This study supports the notion that the electron transport chain is biochemically linked to pilus assembly in A. oris via oxidative folding of pilin precursors

Throughput analysis of non-orthogonal multiple access and orthogonal multiple access assisted wireless energy harvesting K-hop relaying networks

This study introduces the non-orthogonal multiple access (NOMA) technique into the wireless energy harvesting K-hop relay network to increase throughput. The relays have no dedicated energy source and thus depend on energy harvested by wireless from a power beacon (PB). Recently, NOMA has been promoted as a technology with the potential to enhance connectivity, reduce latency, increase fairness amongst users, and raise spectral effectiveness compared to orthogonal multiple access (OMA) technology. For performance considerations, we derive exact throughput expressions for NOMA and OMA-assisted multi-hop relaying and compare the performance between the two. The obtained results are validated via Monte Carlo simulations

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated.IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen

Transcriptome sequencing of the human pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into its transcriptional landscape and into DtxR-mediated transcriptional regulation

Wittchen M, Busche T, Gaspar AH, et al. Transcriptome sequencing of the human pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into its transcriptional landscape and into DtxR-mediated transcriptional regulation. BMC Genomics. 2018;19(1): 82.Background The human pathogen Corynebacterium diphtheriae is the causative agent of diphtheria. In the 1990s a large diphtheria outbreak in Eastern Europe was caused by the strain C. diphtheriae NCTC 13129. Although the genome was sequenced more than a decade ago, not much is known about its transcriptome. Our aim was to use transcriptome sequencing (RNA-Seq) to close this knowledge gap and gain insights into the transcriptional landscape of a C. diphtheriae tox+ strain. Results We applied two different RNA-Seq techniques, one to retrieve 5′-ends of primary transcripts and the other to characterize the whole transcriptional landscape in order to gain insights into various features of the C. diphtheriae NCTC 13129 transcriptome. By examining the data we identified 1656 transcription start sites (TSS), of which 1202 were assigned to genes and 454 to putative novel transcripts. By using the TSS data promoter regions recognized by the housekeeping sigma factor σA and its motifs were analyzed in detail, revealing a well conserved −10 but an only weakly conserved −35 motif, respectively. Furthermore, with the TSS data 5’-UTR lengths were explored. The observed 5’-UTRs range from zero length (leaderless transcripts), which make up 20% of all genes, up to over 450 nt long leaders, which may harbor regulatory functions. The C. diphtheriae transcriptome consists of 471 operons which are further divided into 167 sub-operon structures. In a differential expression analysis approach, we discovered that genetic disruption of the iron-sensing transcription regulator DtxR, which controls expression of diphtheria toxin (DT), causes a strong influence on general gene expression. Nearly 15% of the genome is differentially transcribed, indicating that DtxR might have other regulatory functions in addition to regulation of iron metabolism and DT. Furthermore, our findings shed light on the transcriptional landscape of the DT encoding gene tox and present evidence for two tox antisense RNAs, which point to a new way of transcriptional regulation of toxin production. Conclusions This study presents extensive insights into the transcriptome of C. diphtheriae and provides a basis for future studies regarding gene characterization, transcriptional regulatory networks, and regulation of the tox gene in particular

Evolution of substrate specificity in a retained enzyme driven by gene loss.

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Our results show how gene loss can drive the evolution of substrate specificity from retained enzymes