CI:Mor interactions in the lysogeny switches of Lactococcus lactis TP901-1 and Staphylococcus aureus φ13 bacteriophages.

Aim: To structurally characterize in detail the interactions between the phage repressor (CI) and the antirepressor (Mor) in the lysis-lysogeny switches of two Gram-positive bacteriophages, the lactococcal TP901-1 and staphylococcal φ13. Methods: We use crystallographic structure determination, computational structural modeling, and analysis, as well as biochemical methods, to elucidate similarities and differences in the CI:Mor interactions for the two genetic switches. Results: By comparing a newly determined and other available crystal structures for the N-terminal domain of CI (CI-NTD), we show that the CI interface involved in Mor binding undergoes structural changes upon binding in TP901-1. Most importantly, we show experimentally for the first time the direct interaction between CI and Mor for φ13, and model computationally the interaction interface. The computational modeling supports similar side chain rearrangements in TP901-1 and φ13. Conclusion: This study ascertains experimentally that, like in the TP901-1 lysogeny switch, staphylococcal φ13 CI and Mor interact with each other. The structural basis of the interaction of φ13 CI and Mor was computationally modeled and is similar to the interaction demonstrated experimentally between TP901-1 CI-NTD and Mor, likely involving similar rearrangement of residue side chains during the formation of the complex. The study identifies one CI residue, Glu69, which unusually interacts primarily through its aliphatic chain with an aromatic residue on Mor after changing its conformation compared to the un-complexed structure. This and other residues at the interface are suggested for investigation in future studies.

Minimizing acetate formation from overflow metabolism in Escherichia coli: comparison of genetic engineering strategies to improve robustness toward sugar gradients in large-scale fermentation processes.

Introduction: Escherichia coli, a well characterized workhorse in biotechnology, has been used to produce many recombinant proteins and metabolites, but have a major drawback in its tendency to revert to overflow metabolism. This phenomenon occurs when excess sugar triggers the production of mainly acetate under aerobic conditions, a detrimental by-product that reduces carbon efficiency, increases cell maintenance, and ultimately inhibits growth. Although this can be prevented by controlled feeding of the sugar carbon source to limit its availability, gradients in commercial-scale bioreactors can still induce it in otherwise carbon-limited cells. While the underlying mechanisms have been extensively studied, these have mostly used non-limited cultures. In contrast, industrial production typically employs carbon-limited processes, which results in a substantially different cell physiology. Objective: The objective of this study was to evaluate and compare the efficiency of different metabolic engineering strategies with the aim to reduce overflow metabolism and increase the robustness of an industrial 2'-O-fucosyllactose producing strain under industrially relevant conditions. Methods: Three distinct metabolic engineering strategies were compared: i) alterations to pathways leading to and from acetate, ii) increased flux towards the tricarboxylic acid (TCA) cycle, and iii) reduced glucose uptake rate. The engineered strains were evaluated for growth, acetate formation, and product yield under non-limiting batch conditions, carbon limited fed-batch conditions, and after a glucose pulse in fed-batch mode. Results and Discussion: The findings demonstrated that blockage of the major acetate production pathways by deletion of the pta and poxB genes or increased carbon flux into the TCA cycle by overexpression of the gltA and deletion of the iclR genes, were efficient ways to reduce acetate accumulation. Surprisingly, a reduced glucose uptake rate did not reduce acetate formation despite it having previously been shown as a very effective strategy. Interestingly, overexpression of gltA was the most efficient way to reduce acetate accumulation in non-limited cultures, whereas disruption of the poxB and pta genes was more effective for carbon-limited cultures exposed to a sudden glucose shock. Strains from both strategies showed increased tolerance towards a glucose pulse during carbon-limited growth indicating feasible ways to engineer industrial E. coli strains with enhanced robustness.

The global transcriptomes of Salmonella enterica serovars Gallinarum, Dublin and Enteritidis in the avian host.

Salmonella enterica serovar Gallinarum causes Fowl Typhoid in poultry, and it is host specific to avian species. The reasons why S. Gallinarum is restricted to avians, and at the same time predominately cause systemic infections in these hosts, are unknown. In the current study, we developed a surgical approach to study gene expression inside the peritoneal cavity of hens to shed light on this. Strains of the host specific S. Gallinarum, the cattle-adapted S. Dublin and the broad host range serovar, S. Enteritidis, were enclosed in semi-permeable tubes and surgically placed for 4 h in the peritoneal cavity of hens and for control in a minimal medium at 41.2 °C. Global gene-expression under these conditions was compared between serovars using tiled-micro arrays with probes representing the genome of S. Typhimurium, S. Dublin and S. Gallinarum. Among other genes, genes of SPI-13, SPI-14 and the macrophage survival gene mig-14 were specifically up-regulated in the host specific serovar, S. Gallinarum, and further studies into the role of these genes in host specific infection are highly indicated. Analysis of pathways and GO-terms, which were enriched in the host specific S. Gallinarum without being enriched in the two other serovars indicated that host specificity was characterized by a metabolic fine-tuning as well as unique expression of virulence associated pathways. The cattle adapted serovar S. Dublin differed from the two other serovars by a lack of up-regulation of genes encoded in the virulence associated pathogenicity island 2, and this may explain the inability of this serovar to cause disease in poultry.

Impact of Elevated Levels of Dissolved CO2 on Performance and Proteome Response of an Industrial 2'-Fucosyllactose Producing Escherichia coli Strain.

Large-scale microbial industrial fermentations have significantly higher absolute pressure and dissolved CO2 concentrations than otherwise comparable laboratory-scale processes. Yet the effect of increased dissolved CO2 (dCO2) levels is rarely addressed in the literature. In the current work, we have investigated the impact of industrial levels of dCO2 (measured as the partial pressure of CO2, pCO2) in an Escherichia coli-based fed-batch process producing the human milk oligosaccharide 2'-fucosyllactose (2'-FL). The study evaluated the effect of high pCO2 levels in both carbon-limited (C-limited) and carbon/nitrogen-limited (C/N-limited) fed-batch processes. High-cell density cultures were sparged with 10%, 15%, 20%, or 30% CO2 in the inlet air to cover and exceed the levels observed in the industrial scale process. While the 10% enrichment was estimated to achieve similar or higher pCO2 levels as the large-scale fermentation it did not impact the performance of the process. The product and biomass yields started being affected above 15% CO2 enrichment, while 30% impaired the cultures completely. Quantitative proteomics analysis of the C-limited process showed that 15% CO2 enrichment affected the culture on the protein level, but to a much smaller degree than expected. A more significant impact was seen in the dual C/N limited process, which likely stemmed from the effect pCO2 had on nitrogen availability. The results demonstrated that microbial cultures can be seriously affected by elevated CO2 levels, albeit at higher levels than expected.

Flexible linker modulates the binding affinity of the TP901-1 CI phage repressor to DNA.

Temperate bacteriophages can switch between two life cycles following infection of a host bacterium: the lytic or lysogenic life cycle. The choice between these is controlled by a bistable genetic switch. We investigated the genetic switch of the lactococcal temperate bacteriophage, TP901-1, which is controlled by two regulatory proteins, the Clear 1 (CI) repressor and modulator of repression (MOR) antirepressor. CI consists of a DNA-binding N-terminal domain and a C-terminal domain responsible for oligomerization, connected by a flexible interdomain linker. Full-length CI is hexameric, whereas the truncated version CI with 58 C-terminal residues truncated (CIΔ58), missing the second C-terminal subdomain, is dimeric, but binds with the same affinity as full-length CI to the OL operator site, responsible for lytic genes transcription repression. Three variants of CIΔ58 with shorter, longer, and PP substituted linkers were produced and confirmed by circular dichroism spectroscopy and nanodifferential scanning fluorimetry to be well folded. With small-angle X-ray scattering, we delineated the conformational space sampled by the variants and wild-type in solution and found that shortening and lengthening the linker decrease and increase this, respectively, as also substantiated by molecular dynamics and as intended. Isoelectric focusing electrophoresis confirmed that all variants are able to bind to the MOR antirepressor. However, using electrophoretic mobility shift assays, we showed that shortening and lengthening the linker lead to a 94 and 17 times decrease in affinity to OL , respectively. Thus, an appropriate linker length appears to be crucial for appropriate DNA-binding and subsequent TP901-1 genetic switch function.

Characterization of the genetic switch from phage ɸ13 important for Staphylococcus aureus colonization in humans.

Temperate phages are bacterial viruses that after infection either reside integrated into a bacterial genome as prophages forming lysogens or multiply in a lytic lifecycle. The decision between lifestyles is determined by a switch involving a phage-encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here, we investigate the switch of phage ɸ13 from the human pathogen Staphylococcus aureus. ɸ13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the ɸ13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high-affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from ɸ13 with the lytic promoter fused to lacZ into S. aureus and Bacillus subtilis. Analysis of ß-galactosidase expression indicated that decision frequency is independent of host factors. The white "lysogenic" phenotype, which relies on the expression of cI, could be switched to a stable blue "lytic" phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage ɸ13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus.

Revealing the mechanism of repressor inactivation during switching of a temperate bacteriophage.

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.

Repression of the lysogenic PR promoter in bacteriophage TP901-1 through binding of a CI-MOR complex to a composite OM-OR operator.

A functional genetic switch from the lactococcal bacteriophage TP901-1, deciding which of two divergently transcribing promoters becomes most active and allows this bi-stable decision to be inherited in future generations requires a DNA region of less than 1 kb. The fragment encodes two repressors, CI and MOR, transcribed from the PR and PL promoters respectively. CI can repress the transcription of the mor gene at three operator sites (OR, OL, and OD), leading to the immune state. Repression of the cI gene, leading to the lytic (anti-immune) state, requires interaction between CI and MOR by an unknown mechanism, but involving a CI:MOR complex. A consensus for putative MOR binding sites (OM sites), and a common topology of three OM sites adjacent to the OR motif was here identified in diverse phage switches that encode CI and MOR homologs, in a search for DNA sequences similar to the TP901-1 switch. The OR site and all putative OM sites are important for establishment of the anti-immune repression of PR, and a putative DNA binding motif in MOR is needed for establishment of the anti-immune state. Direct evidence for binding between CI and MOR is here shown by pull-down experiments, chemical crosslinking, and size exclusion chromatography. The results are consistent with two possible models for establishment of the anti-immune repression of cI expression at the PR promoter.

Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase.

BACKGROUND: PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. RESULTS: In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. CONCLUSIONS: In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA-amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.

Influence of chlorides and phosphates on the antiadhesive, antibacterial, and electrochemical properties of an electroplated copper-silver alloy.

Antimicrobial surfaces such as copper alloys can reduce the spread of pathogenic microorganisms, e.g., in healthcare settings; however, the surface chemistry and thus the antibacterial activity are influenced by environmental parameters such as cleaning and disinfection procedures. Therefore, the purpose of the present study was to assess how copper-complexing compounds (chlorides and phosphates), common to the clinical environment, can affect the surface chemistry and the antiadhesive and antibacterial properties of a newly developed antibacterial copper-silver alloy and the single alloying metals. The authors demonstrated that the antiadhesion efficacy against S. aureus 8325 was the highest when the copper-silver alloy and copper surfaces (four- and two-log bacterial reduction compared to stainless steel controls, respectively) were exposed to chloride-containing suspensions. This was explained by the electrochemical activity of copper that dissolved as Cu+, highly toxic to the bacterial cells, in the presence of Cl- and eventually formed a chlorine- and oxygen-rich layer with the incorporation of phosphorus, if also phosphates were present. If chlorides were omitted from the wet environment, there was no difference (P > 0.05) in bacterial counts on copper-silver alloy, copper, silver, and AISI 316 stainless steel control surfaces, due to the fact that no oxidizing conditions were established and therefore there was no dissolution of copper ions from copper-silver alloy and copper surfaces. However, under dry conditions, copper-silver alloy and pure copper surfaces were antibacterial also in the absence of chlorides, suggesting a marked difference between dry and wet conditions in terms of the interactions between surfaces and bacteria. The authors conclude that an attentive design of control policies integrating disinfection interventions and antimicrobial surfaces, such as the copper-silver alloy coating, can be a beneficial solution in fighting the spread of antibiotic resistant bacterial strains and potentially reducing the number of disease outbreaks.

Basics of Antibody Phage Display Technology.

Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.

Linear peptidomimetics as potent antagonists of Staphylococcus aureus agr quorum sensing.

Staphylococcus aureus is an important pathogen causing infections in humans and animals. Increasing problems with antimicrobial resistance has prompted the development of alternative treatment strategies, including antivirulence approaches targeting virulence regulation such as the agr quorum sensing system. agr is naturally induced by cyclic auto-inducing peptides (AIPs) binding to the AgrC receptor and cyclic peptide inhibitors have been identified competing with AIP binding to AgrC. Here, we disclose that small, linear peptidomimetics can act as specific and potent inhibitors of the S. aureus agr system via intercepting AIP-AgrC signal interaction at low micromolar concentrations. The corresponding linear peptide did not have this ability. This is the first report of a linear peptide-like molecule that interferes with agr activation by competitive binding to AgrC. Prospectively, these peptidomimetics may be valuable starting scaffolds for the development of new inhibitors of staphylococcal quorum sensing and virulence gene expression.

Metagenomic Analysis of Therapeutic PYO Phage Cocktails from 1997 to 2014.

Phage therapy has regained interest in recent years due to the alarming spread of antibiotic resistance. Whilst phage cocktails are commonly sold in pharmacies in countries such as Georgia and Russia, this is not the case in western countries due to western regulatory agencies requiring a thorough characterization of the drug. Here, DNA sequencing of constituent biological entities constitutes a first step. The pyophage (PYO) cocktail is one of the main commercial products of the Georgian Eliava Institute of Bacteriophage, Microbiology and Virology and is used to cure skin infections. Since its first production in the 1930s, the composition of the cocktail has been periodically modified to add phages effective against emerging pathogenic strains. In this paper, we compared the composition of three PYO cocktails from 1997 (PYO97), 2000 (PYO2000) and 2014 (PYO2014). Based on next generation sequencing, de novo assembly and binning of contigs into draft genomes based on tetranucleotide distance, thirty and twenty-nine phage draft genomes were predicted in PYO97 and PYO2014, respectively. Of these, thirteen and fifteen shared high similarity to known phages. Eleven draft genomes were found to be common in the two cocktails. One of these showed no similarity to publicly available phage genomes. Representatives of phages targeting E. faecalis, E. faecium, E. coli, Proteus, P. aeruginosa and S. aureus were found in both cocktails. Finally, we estimated larger overlap of the PYO2000 cocktail to PYO97 compared to PYO2014. Using next generation sequencing and metagenomics analysis, we were able to characterize and compare the content of PYO cocktails separated by 17 years in time. Even though the cocktail composition is upgraded every six months, we found it to remain relatively stable over the years.

Butanol is cytotoxic to Lactococcus lactis while ethanol and hexanol are cytostatic.

Lactic acid bacteria currently used extensively by the dairy industry have a superior tolerance towards short-chain alcohols, which makes them interesting targets for use in future bio-refineries. The mechanism underlying the alcohol tolerance of lactic acid bacteria has so far received little attention. In the present study, the physiological alcohol stress response of Lactococcus lactis subsp. cremoris MG1363 towards the primary, even-chain alcohols ethanol, butanol and hexanol, was characterized. The alcohol tolerance of L. lactis was found to be comparable to those reported for highly alcohol-resistant lactic acid bacteria. Combined results from alcohol survival rate, live/dead staining, and a novel usage of the ß-galactosidase assay, revealed that while high concentrations of ethanol and hexanol were cytostatic to L. lactis, high concentrations of butanol were cytotoxic, causing irreparable damages to the cell membrane.

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance.

Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

Production of the Bioactive Compounds Violacein and Indolmycin Is Conditional in a maeA Mutant of Pseudoalteromonas luteoviolacea S4054 Lacking the Malic Enzyme.

It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7-10% the amount of violacein compared to the wild type strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wild type. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea.

Ribosomal dimerization factor YfiA is the major protein synthesized after abrupt glucose depletion in Lactococcus lactis.

We analysed the response of the model bacterium Lactococcus lactis to abrupt depletion of glucose after several generations of exponential growth. Glucose depletion resulted in a drastic drop in the energy charge accompanied by an extremely low GTP level and an almost total arrest of protein synthesis. Strikingly, the cell prioritized the continued synthesis of a few proteins, of which the ribosomal dimerization factor YfiA was the most highly expressed. Transcriptome analysis showed no immediate decrease in total mRNA levels despite the lowered nucleotide pools and only marginally increased levels of the yfiA transcript. Severe up-regulation of genes in the FruR, CcpA, ArgR and AhrC regulons were consistent with a downshift in carbon and energy source. Based upon the results, we suggest that transcription proceeded long enough to record the transcriptome changes from activation of the FruR, CcpA, ArgR and AhrC regulons, while protein synthesis stopped due to an extremely low GTP concentration emerging a few minutes after glucose depletion. The yfiA deletion mutant exhibited a longer lag phase upon replenishment of glucose and a faster death rate after prolonged starvation supporting that YfiA-mediated ribosomal dimerization is important for keeping long-term starved cells viable and competent for growth initiation.

Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1.

The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CI∆58, forms dimers. We identify the dimerization region of CI∆58 as CTD1 and determine its secondary structure to be helical both within the context of CI∆58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CI∆58 interacts with the OL operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CI∆58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.

Clear Plaque Mutants of Lactococcal Phage TP901-1.

We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1.

The In Vitro Redundant Enzymes PurN and PurT Are Both Essential for Systemic Infection of Mice in Salmonella enterica Serovar Typhimurium.

Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundant in vitro in Salmonella enterica serovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that single-gene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ΔpurT mutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ΔpurN mutant was reduced approximately 50% in 20 h. The attenuation of the ΔpurT mutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C1 units in vivo (CI = 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C1 units in vitro The results with the ΔgcvT mutant further revealed that formation of serine by SerA and further conversion of serine into C1 units and glycine by GlyA were not sufficient to ensure C1 formation in S Typhimurium in vivo The results of the present study call for reinvestigations of the concept of metabolic redundancy in S Typhimurium in vivo.