Rhodobacter capsulatus DprA is essential for RecA‐mediated gene transfer agent (RcGTA) recipient capability regulated by quorum‐sensing and the CtrA response regulator

Gene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best‐studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA‐mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA‐protecting protein A) homologue is essential for RcGTA‐mediated gene acquisition, and that dprA expression is induced by gtaI‐dependent quorum‐sensing and non‐phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C‐terminal region that resembles a dsDNA‐binding protein domain. Purified His‐tagged R. capsulatus DprA protein bound to both single‐stranded (ss)DNA and double‐stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single‐cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.     

A Clostridium perfringens hem gene cluster contains a cysG(B) homologue that is involved in cobalamin biosynthesis

The hem gene cluster, which consists of hemA, cysG(B), hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl-tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N-terminal region of C. perfringens HemD is homologous to those reported for the C-terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C. perfringens CysG(B) is a predicted 220-residue protein which shows homology to the N-terminal region of S. typhimurium CysG. Disruption of the cysG(B) gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12-deficient medium, the mutant strain showed a four-fold increase in its doubling time compared with that of the wild-type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysG(B) is involved in the chelation of cobalt to precorrin II as suggested for the CysG(B) domain of S. typhimurium CysG, enabling the synthesis of cobalamin.     

Streptococcus pneumoniae PBP2x mid‐cell localization requires the C‐terminal PASTA domains and is essential for cell shape maintenance

The transpeptidase activity of the essential penicillin‐binding protein 2x (PBP2x) of Streptococcus pneumoniae is believed to be important for murein biosynthesis required for cell division. To study the molecular mechanism driving localization of PBP2x in live cells, we constructed a set of N‐terminal GFP–PBP2x fusions under the control of a zinc‐inducible promoter. The ectopic fusion protein localized at mid‐cell. Cells showed no growth defects even in the absence of the genomic pbp2x, demonstrating that GFP–PBP2x is functional. Depletion of GFP–PBP2x resulted in severe morphological alterations, confirming the essentiality of PBP2x and demonstrating that PBP2x is required for cell division and not for cell elongation. A genetically or antibiotic inactivated GFP–PBP2x still localized at septal sites. Remarkably, the same was true for a GFP–PBP2x derivative containing a deletion of the central transpeptidase domain, although only in the absence of the protease/chaperone HtrA. Thus localization is independent of the catalytic transpeptidase domain but requires the C‐terminal PASTA domains, identifying HtrA as targeting GFP–PBP2x derivatives. Finally, PBP2x was positioned at the septum similar to PBP1a and the PASTA domain containing StkP protein, confirming that PBP2x is a key element of the divisome complex.     

A putative gene cluster from a Lyngbya wollei bloom that encodes paralytic shellfish toxin biosynthesis

Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds.     

Gene cluster for biosynthesis of thermophilin 1277--a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide

Aims: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. Methods and Results: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer‐walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9‐kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI‐expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. Conclusions: Thermophilin 1277 biosynthesis genes were encoded by a 9·9‐kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. Significance and Impact of the Study: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.     

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium Salmonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.     

A food‐grade site‐directed mutagenesis system for Streptococcus thermophilus LMG 18311

Aims: To develop a general method for site‐directed mutagenesis in the dairy starter strain Streptococcus thermophilus LMG 18311 which does not depend on antibiotic‐resistance genes or other selection markers for the identification of transformants. Methods and Results: In a previous study, we demonstrated that Strep. thermophilus LMG 18311 can be made competent for natural genetic transformation by overexpression of the alternative sigma factor ComX. In the present study, we wanted to investigate whether the natural transformation mechanism of Strep. thermophilus LMG 18311 is efficient enough to make it feasible to perform site‐directed mutagenesis in this strain without the use of a selection marker. Competent bacteria were mixed with a DNA fragment engineered to contain a nonsense and a frameshift mutation in the middle of the target gene (lacZ) and subsequently seeded on agar plates. By performing colony‐lift hybridization using a digoxigenin‐labelled oligonucleotide probe, we succeeded in identifying transformants containing the sought after mutation. Conclusions: By exploiting the natural transformability of Strep. thermophilus LMG 18311 and standard molecular methods, we have demonstrated that the genome of this bacterium can be altered at preselected sites without introduction of any foreign DNA. Significance and Impact of the Study: A food‐grade site‐directed mutagenesis system has been developed for Strep. thermophilus LMG 18311 that can be used by the dairy industry to construct starter strains with novel and/or improved properties.     

Quorum‐sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA)

The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteriophage that packages almost random genomic DNA segments that may be transferred to other R. capsulatus cells. Gene transfer agents are produced by a number of prokaryotes; however, no receptors have been identified. We investigated the RcGTA recipient capability of wild‐type R. capsulatus cells at different culture growth phases, and found that the frequency of RcGTA‐dependent acquisition of an allele increases as cultures enter the stationary phase. We also found that RcGTA adsorption to cells follows a similar trend. RcGTA recipient capability and adsorption were found to be dependent on the GtaR/I quorum‐sensing (QS) system. Production of an extracellular polysaccharide was found to be regulated by GtaR/I QS, as was production of the cell capsule. A number of QS‐regulated putative polysaccharide biosynthesis genes were identified, and mutagenesis of two of these genes, rcc01081 and rcc01932, yielded strains that lack a capsule. Furthermore, these mutants were impaired in RcGTA recipient capability and adsorption, as was a non‐encapsulated wild‐type isolate of R. capsulatus. Overall, our results indicate that capsular polysaccharide is a receptor for the gene transfer agent of R. capsulatus, RcGTA.     

Cloning of the gene cluster responsible for biosynthesis of KS-505a (longestin), a unique tetraterpenoid

KS-505a (longestin), produced by Streptomyces argenteolus, has a unique structure that consists of a tetraterpene (C40) skeleton, to which a 2-O-methylglucuronic acid and an o-succinyl benzoate moiety are attached. It is a novel inhibitor of calmodulin-dependent cyclic-nucleotide phosphodiesterase, which is representative of a potent anti-amnesia drug. As a first step to understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we cloned a KS505a biosynthetic gene cluster. First we searched for a gene encoding octaprenyl diphosphates, which yielded a C40 precursor by PCR, and four candidate genes were obtained. Among these, one was confirmed to have the expected enzyme activity by recombinant enzyme assay. On the basis of an analysis of the flanking regions of the gene, a putative KS-505a biosynthetic gene cluster consisting of 24 ORFs was judged perhaps to be present on a 28-kb DNA fragment. A gene disruption experiment was also employed to confirm that the cluster indeed participated in KS-505a biosynthesis. This is believed to be the first report detailing the gene cluster of a cyclized tetraterpenoid.     

Structure and function of LGP2, a DEX(D/H) helicase that regulates the innate immunity response

RNA recognition receptors are important for detection of and response to viral infections. RIG-I and MDA5 are cytoplasmic DEX(D/H) helicase proteins that can induce signaling in response to RNA ligands, including those from viral infections. LGP2, a homolog of RIG-I and MDA5 without the caspase recruitment domain required for signaling, plays an important role in modulating signaling by MDA5 and RIG-I, presumably through heterocomplex formation and/or by serving as a sink for RNAs. Here we demonstrate that LGP2 can be coexpressed with RIG-I to inhibit activation of the NF-kappaB reporter expression and that LGP2 protein produced in insect cells can bind both single- and double-stranded RNA (dsRNA), with higher affinity and cooperativity for dsRNA. Electron microscopy and image reconstruction were used to determine the shape of the LGP2 monomer in the absence of dsRNA and of the dimer complexed to a 27-bp dsRNA. LGP2 has striking structural similarity to the helicase domain of the superfamily 2 DNA helicase, Hef.     

Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross‐talk in Streptococcus pneumoniae D39 cells

WalRK (YycFG) two‐component systems (TCSs) of low‐GC Gram‐positive bacteria play critical roles in regulating peptidogylcan hydrolase genes involved in cell division and wall stress responses. The WalRK (VicRK) TCSs of Streptococcus pneumoniae (pneumococcus) and other Streptococcus species show numerous differences with those of other low‐GC species. Notably, the pneumococcal WalK sensor kinase is not essential for normal growth in culture, unlike its homologues in Bacillus and Staphylococcus species. The WalK sensor kinase possesses histidine autokinase activity and mediates dephosphorylation of phosphorylated WalR～P response regulator. To understand the contributions of these two WalK activities to pneumococcal growth, we constructed and characterized a set of walK kinase and phosphatase mutants in biochemical reactions and in cells. We identified an amino acid substitution in WalK that significantly reduces phosphatase activity, but not other activities. Comparisons were made between WalRK regulon expression levels and WalR～P amounts in cells determined by Phos‐tag SDS‐PAGE. Reduction of WalK phosphatase activity resulted in nearly 90% phosphorylation to WalR～P, consistent with the conclusion that WalK phosphatase is strongly active in exponentially growing cells. WalK phosphatase activity was also shown to depend on the WalK PAS domain and to limit cross‐talk and the recovery of WalR～P from walK⁺ cells.     

Developing a strong anion exchange/RP (SAX/RP) 2D LC system for high‐abundance proteins depletion in human plasma

Human plasma is dominated by high‐abundance proteins which severely impede the detection of low‐abundance proteins. Unfortunately, now there is no efficient method for large‐scale depletion of high‐abundance proteins in human plasma. In this study, we developed a new strategy, strong anion exchange (SAX)/RP 2D LC system, which has potential for large‐scale depletion of high‐abundance proteins in human plasma. Separation gradients of the system were optimized to ensure an extensive separation of plasma proteins. Plasma was fractionated into 67 fractions by SAX. All these fractions were subjected a thorough separation by the 2D RPLC and 66 peaks with high UV absorption (>20 mAU) at 215 nm were collected. Proteins in these peaks were identified by LC‐MS/MS analysis. Results showed that 83 proteins could be identified in these peaks, 68 among them were reported to be high‐ or middle‐abundance proteins in plasma. All these proteins had definite retention times and were mapped in the 2D SAX‐RP system, which resulted in accurate depletion of high‐abundance proteins with ease. Our studies provide a convenient and effective method for large‐scale depletion of high‐abundance proteins and in‐depth research in human plasma proteomics.