Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization.

BACKGROUND: This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections. METHODS: KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10. RESULTS: KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family. CONCLUSION: The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.

Investigates the ability of plant extracts from Lens culinaris to protect zucchini from the Zucchini yellow mosaic virus (ZYMV).

Evaluate the impact of extracts from the Lens culinaris plant on a number of physiological and biochemical parameters in squash leaves infected with ZYMV in this work. Compared to the untreated leaves, ZYMV infected leaves showed a range of symptoms, such as severe mosaic, size reduction, stunting, and deformation. Analysis of physiological data revealed that L. culinaris extract lectin therapies and viral infections had an impact on metabolism. Protein, carbohydrate, and pigment levels were all lowered by viral infection. However, phenolic compounds, total protein, total carbohydrates, total amino acids, proline, total chlorophyll and peroxidases levels are considerably elevated with all extract therapies. The other biochemical parameters also displayed a variety of changes. Moreover shoot length, number of leaves and number of flowers was significantly increased compared to viral control in all treatments. The L. culinaris extract treatment increases the plant's ZYMV resistance. This is detectable through reduction of the plants treated with lentil lectin pre and post virus inoculation, reduction in disease severity and viral concentration, and percentage of the infected plants has a virus. All findings demonstrate significant metabolic alterations brought by viral infections or L. culinaris extract treatments, and they also suggest that exogenous extract treatments is essential for activating the body's defences against ZYMV infection.

Hyaluronic acid production by Klebsiella pneumoniae strain H15 (OP354286) under different fermentation conditions.

BACKGROUND: Hyaluronic acid (HA) has gained significant attention due to its unique physical, chemical, and biological properties, making it widely used in various industries. This study aimed to screen bacterial isolates for HA production, characterize favorable fermentation conditions, and evaluate the inhibitory effect of bacterial HA on cancer cell lines. RESULTS: A total of 108 bacterial isolates from diverse sources were screened for HA production using HPLC, turbidimetric, and carbazole determination methods. Among the HA-producing isolates, Klebsiella pneumoniae H15 isolated from an animal feces sample, was superior in HA production. The strain was characterized based on its morphological, cultural, and biochemical characteristics. Molecular identification using 16S rDNA sequencing and phylogenetic analysis confirmed its identity. Fermentation conditions, including pH, temperature, time, and agitation rate, were optimized to maximize HA production. The basal medium, comprising sucrose (7.0%) as carbon source and combined yeast extract with peptone (1.25% each) as nitrogen substrate, favored the highest HA production at pH 8.0, for 30 h, at 30 °C, under shaking at 180 rpm. The average maximized HA concentration reached 1.5 g L-1. Furthermore, bacterial HA exhibited a significant inhibitory effect on three cancer cell lines (MCF-7, HepG-2 and HCT), with the lowest concentration ranging from 0.98-3.91 µg mL-1. CONCLUSIONS: K. pneumoniae H15, isolated from animal feces demonstrated promising potential for HA production. The most favorable fermentation conditions led to a high HA production. The inhibitory effect of bacterial HA on cancer cell lines highlights its potential therapeutic applications. These findings contribute to a broader understanding and utilization of HA in various industries and therapeutic applications.

Detection of multidrug-resistant Shiga toxin-producing Escherichia coli in some food products and cattle faeces in Al-Sharkia, Egypt: one health menace.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen, that is transmitted from a variety of animals, especially cattle to humans via contaminated food, water, feaces or contact with infected environment or animals. The ability of STEC strains to cause gastrointestinal complications in human is due to the production of Shiga toxins (sxt). However, the transmission of multidrug-resistance STEC strains are linked with a severity of disease outcomes and horizontal spread of resistance genes in other pathogens. The result of this has emerged as a significant threat to public health, animal health, food safety, and the environment. Therefore, the purpose of this study is to investigate the antibiogram profile of enteric E. coli O157 isolated from food products and cattle faeces samples in Zagazig City, Al-Sharkia, Egypt, and to reveal the presence of Shiga toxin genes stx1 and stx2 as virulence factors in multidrug-resistant isolates. In addition to this, the partial 16S rRNA sequencing was used for the identification and genetic recoding of the obtained STEC isolates. RESULTS: There was a total of sixty-five samples collected from different geographical regions at Zagazig City, Al-Sharkia-Egypt, which were divided into: 15 chicken meat (C), 10 luncheon (L), 10 hamburgers (H), and 30 cattle faeces (CF). From the sixty-five samples, only 10 samples (one from H, and 9 from CF) were identified as suspicious E. coli O157 with colourless colonies on sorbitol MacConkey agar media with Cefixime- Telurite supplement at the last step of most probable number (MPN) technique. Eight isolates (all from CF) were identified as multidrug-resistant (MDR) as they showed resistance to three antibiotics with multiple antibiotic resistance (MAR) index ≥ 0.23, which were assessed by standard Kirby-Bauer disc diffusion method. These eight isolates demonstrated complete resistance (100%) against amoxicillin/clavulanic acid, and high frequencies of resistance (90%, 70%, 60%,60%, and 40%) against cefoxitin, polymixin, erythromycin, ceftazidime, and piperacillin, respectively. Those eight MDR E. coli O157 underwent serological assay to confirm their serotype. Only two isolates (CF8, and CF13), both from CF, were showed strong agglutination with antisera O157 and H7, as well as resistance against 8 out of 13 of the used antibiotics with the highest MAR index (0.62). The presence of virulence genes Shiga toxins (stx1 and stx2) was assessed by PCR technique. CF8 was confirmed for carrying stx2, while CF13 was carrying both genes stx1, and stx2. Both isolates were identified by partial molecular 16S rRNA sequencing and have an accession number (Acc. No.) of LC666912, and LC666913 on gene bank. Phylogenetic analysis showed that CF8, and CF13 were highly homologous (98%) to E. coli H7 strain, and (100%) to E. coli DH7, respectively. CONCLUSION: The results of this study provides evidence for the occurrence of E. coli O157:H7 that carries Shiga toxins stx1 and/or stx2, with a high frequency of resistance to antibiotics commonly used in human and veterinary medicine, in Zagazig City, Al-Sharkia, Egypt. This has a high extent of public health risk posed by animal reservoirs and food products with respect to easy transmission causing outbreaks and transfer resistance genes to other pathogens in animal, human, and plants. Therefore, environmental, animal husbandry, and food product surveillance, as well as, clinical infection control, must be strengthened to avoid the extra spread of MDR pathogens, especially MDR STEC strains.

Antibiotic Resistance, Virulence Gene Detection, and Biofilm Formation in Aeromonas spp. Isolated from Fish and Humans in Egypt.

The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. The aim of the current study was to determine the prevalence of Aeromonas spp. in raw fish markets and humans in Zagazig, Egypt; identify the factors that contribute to virulence; determine the isolates' profile of antibiotic resistance; and to elucidate the ability of Aeromonas spp. to form biofilms. The examined samples included fish tissues and organs from tilapia (Oreochromis niloticus, n = 160) and mugil (Mugil cephalus, n = 105), and human skin swabs (n = 51) and fecal samples (n = 27). Based on biochemical and PCR assays, 11 isolates (3.2%) were confirmed as Aeromonas spp. and four isolates (1.2%) were confirmed as A. hydrophila. The virulence genes including haemolysin (hyl A) and aerolysin (aer) were detected using PCR in A. hydrophila in percentages of 25% and 50%, respectively. The antimicrobial resistance of Aeromonas spp. was assessed against 14 antibiotics comprising six classes. The resistance to cefixime (81.8%) and tobramycin (45.4%) was observed. The multiple antibiotic resistance (MAR) index ranged between 0.142-0.642 with 64.2% of the isolates having MAR values equal to 0.642. Biofilm formation capacity was assessed using a microtiter plate assay, and two isolates (18.1%) were classified as biofilm producers. This study establishes a baseline for monitoring and controlling the multidrug-resistant Aeromonas spp. and especially A. hydrophila in marine foods consumed in our country to protect humans and animals.

Metabolic adjustments in response to ATP spilling by the small DX protein in a Streptomyces strain.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms. However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces, the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong ErmE promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health.

Pseudomonas Phage ZCPS1 Endolysin as a Potential Therapeutic Agent.

The challenge of antibiotic resistance has gained much attention in recent years due to the rapid emergence of resistant bacteria infecting humans and risking industries. Thus, alternatives to antibiotics are being actively searched for. In this regard, bacteriophages and their enzymes, such as endolysins, are a very attractive alternative. Endolysins are the lytic enzymes, which are produced during the late phase of the lytic bacteriophage replication cycle to target the bacterial cell walls for progeny release. Here, we cloned, expressed, and purified LysZC1 endolysin from Pseudomonas phage ZCPS1. The structural alignment, molecular dynamic simulation, and CD studies suggested LysZC1 to be majorly helical, which is highly similar to various phage-encoded lysozymes with glycoside hydrolase activity. Our endpoint turbidity reduction assay displayed the lytic activity against various Gram-positive and Gram-negative pathogens. Although in synergism with EDTA, LysZC1 demonstrated significant activity against Gram-negative pathogens, it demonstrated the highest activity against Bacillus cereus. Moreover, LysZC1 was able to reduce the numbers of logarithmic-phase B. cereus by more than 2 log10 CFU/mL in 1 h and also acted on the stationary-phase culture. Remarkably, LysZC1 presented exceptional thermal stability, pH tolerance, and storage conditions, as it maintained the antibacterial activity against its host after nearly one year of storage at 4 °C and after being heated at temperatures as high as 100 °C for 10 min. Our data suggest that LysZC1 is a potential candidate as a therapeutic agent against bacterial infection and an antibacterial bio-control tool in food preservation technology.

Bacteriophages to Control Multi-Drug Resistant Enterococcus faecalis Infection of Dental Root Canals.

Phage therapy is an alternative treatment to antibiotics that can overcome multi-drug resistant bacteria. In this study, we aimed to isolate and characterize lytic bacteriophages targeted against Enterococcus faecalis isolated from root canal infections obtained from clinics at the Faculty of Dentistry, Ismalia, Egypt. Bacteriophage, vB_ZEFP, was isolated from concentrated wastewater collected from hospital sewage. Morphological and genomic analysis revealed that the phage belongs to the Podoviridae family with a linear double-stranded DNA genome, consisting of 18,454, with a G + C content of 32.8%. Host range analysis revealed the phage could infect 10 of 13 E. faecalis isolates exhibiting a range of antibiotic resistances recovered from infected root canals with efficiency of plating values above 0.5. One-step growth curves of this phage showed that it has a burst size of 110 PFU per infected cell, with a latent period of 10 min. The lytic activity of this phage against E. faecalis biofilms showed that the phage was able to control the growth of E. faecalis in vitro. Phage vB_ZEFP could also prevent ex-vivo E. faecalis root canal infection. These results suggest that phage vB_ZEFP has potential for application in phage therapy and specifically in the prevention of infection after root canal treatment.

In vitro characterization of the site-specific recombination system based on genus Habenivirus ϕRSM small serine integrase.

The genus Habenivirus which includes Ralstonia virus ϕRSM encodes a site-specific integrase of a small serine recombinase belonging to the resolvase/invertase family. Here we describe the integrative/excisive recombination reactions mediated by ϕRSM integrase using in vitro assays. The products of attP/attB recombination, i.e. attL and attR, were exactly identical to those found in the prophage ϕRSM in R. solanacearum strains. The minimum size of attB required for integration was determined to be 37 bp, containing a 13 bp core and flanking sequences of 4 bp on the left and 20 bp on the right. ϕRSM integrative recombination proceeds efficiently in vitro in the absence of additional proteins or high-energy cofactors. Excision of a functional phage genome from a prophage fragment was demonstrated in vitro, demonstrating two-way activity of ϕRSM1 integrase. This is the first example of a small serine recombinase from the resolvase/invertase group that functions in integrative and excisive recombination for filamentous phages. This serine integrase could be used as a tool for several genome engineering applications.

Methicillin- and Vancomycin-Resistant Staphylococcus aureus From Humans and Ready-To-Eat Meat: Characterization of Antimicrobial Resistance and Biofilm Formation Ability.

Methicillin-resistant and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) are zoonotic life-threatening pathogens, and their presence in food raises a public health concern. Yet, scarce data are available regarding MRSA and VRSA in both ready-to-eat (RTE) meat and food handlers. This study was undertaken to determine the frequency, antimicrobial resistance, and biofilm-forming ability of MRSA and VRSA isolated from RTE meat (shawarma and burger) and humans (food handlers, and hospitalized patients) in Zagazig city, Sharkia Governorate, Egypt. We analyzed 176 samples (112 human samples: 72 from hospitalized patients and 40 from food handlers, 64 RTE meat samples: 38 from shawarma and 26 from burger). Using phenotypic, PCR-based identification of nuc gene and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 60 coagulase-positive S. aureus (COPS) isolates were identified in the samples as follow: RTE meat (15/64, 23.4%), hospitalized patients (33/72, 45.8%) and food handlers (12/40, 30%). All the COPS isolates were mecA positive (and thus were classified as MRSA) and multidrug resistant with multiple antibiotic resistance indices ranging from 0.25 to 0.92. Overall, resistance to cefepime (96.7%), penicillin (88.3%), were common, followed by ampicillin-sulbactam (65%), ciprofloxacin (55%), nitrofurontoin (51.7%), and gentamicin (43.3%). VRSA was detected in 30.3% of COPS hospitalized patient's isolates, 26.7% of COPS RTE meat isolates and 25% of COPS food handler's isolates. VanA, vanB, or both genes were detected in 64.7, 5.9, and 29.4% of all VAN-resistant isolates, respectively. The majority of the COPS isolates (50/60, 83.3%) have biofilm formation ability and harbored icaA (76%), icaD (74%), icaC (50%), and icaB (46%) biofilm-forming genes. The bap gene was not detected in any of the isolates. The ability of MRSA and VRSA isolates to produce biofilms in addition to being resistant to antimicrobials highlight the danger posed by these potentially virulent microorganisms persisting in RTE meat, food handlers, and patients. Taken together, good hygiene practices and antimicrobial surveillance plans should be strictly implemented along the food chain to reduce the risk of colonization and dissemination of MRSA and VRSA biofilm-producing strains.

Characterization of φEf-vB1 prophage infecting oral Enterococcus faecalis and enhancing bacterial biofilm formation.

Introduction. Enterococcus faecalis is a facultative, anaerobic, opportunistic pathogen associated with medical and dental diseases. Bacterial phenotypic traits and pathogenesis are often influenced by lysogeny.Aim. The aim of this study was to characterize both the morphology and complete genome sequences of induced prophages purified from E. faecalis clinical isolates.Methodology. E. faecalis isolates were recovered from the roots of teeth of patients attending an endodontic clinic. The morphological features of isolated phage were characterized using transmission electron microscopy (TEM). DNA sequencing was performed using the Illumina MiSeq platform.Results. TEM indicated that the isolated φEf-vB1 prophage belongs to the family Siphoviridae. The φEf-vB1 prophage was stable over a wide range of temperatures and pH. Sequencing of φEf-vB1 DNA revealed that the phage genome is 37 561 bp in length with a G+C content of 37.6mol% and contained 53 ORFs. Comparison with previously predicted prophage genomes using blast revealed that φEf-vB1 has a high sequence similarity to previously characterized phage genomes. The lysogenic E. faecalis strain exhibited a higher biofilm formation capacity relative to the non-lysogenic strain.Conclusion. The current findings highlight the role of lysogeny in modification of E. faecalis properties and reveal the potential importance of prophages in E. faecalis biology and pathogenesis.

Correction to: Full genome sequence of a polyvalent bacteriophage infecting strains of Shigella, Salmonella, and Escherichia.

The original version of this article unfortunately contained a mistake.

Full genome sequence of a polyvalent bacteriophage infecting strains of Shigella, Salmonella, and Escherichia.

A novel lytic bacteriophage, Escherichia phage EcS1, was isolated from sewage samples collected in Higashi-Hiroshima, Japan. The complete genome sequence of EcS1 was determined using the Illumina Miseq System. The whole genome of EcS1 was found to be 175,437 bp in length with a mean G+C content of 37.8%. A total of 295 genes were identified as structural, functional, and hypothetical genes. BLAST analysis of the EcS1 genomic sequence revealed the highest identity (79%; query cover of 73-74%) to three T4-related phages that infect Serratia sp. ATCC 39006. Host range experiments revealed that EcS1 has lytic effects on three pathogenic strains of Shigella spp. and a pathogenic strain of Salmonella enterica as well as on E. coli strains. However, two strains of Serratia marcescens showed resistance to this phage. Phylogenetic trees for phage tail fiber protein sequences revealed that EcS1 is closely related to Enterobacteriaceae-infecting phages. Thus, EcS1 is a novel phage that infects several pathogenic strains of the family Enterobacteriaceae.

Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes.

Xanthomonas virus (phage) XacN1 is a novel jumbo myovirus infecting Xanthomonas citri, the causative agent of Asian citrus canker. Its linear 384,670 bp double-stranded DNA genome encodes 592 proteins and presents the longest (66 kbp) direct terminal repeats (DTRs) among sequenced viral genomes. The DTRs harbor 56 tRNA genes, which correspond to all 20 amino acids and represent the largest number of tRNA genes reported in a viral genome. Codon usage analysis revealed a propensity for the phage encoded tRNAs to target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes and seven additional translation-related genes as well as a chaperonin gene found in the XacN1 genome suggests a relative independence of phage replication on host molecular machinery, leading to a prediction of a wide host range for this jumbo phage. We confirmed the prediction by showing a wider host range of XacN1 than other X. citri phages in an infection test against a panel of host strains. Phylogenetic analyses revealed a clade of phages composed of XacN1 and ten other jumbo phages, indicating an evolutionary stable large genome size for this group of phages.

Isolation and characterization of polyvalent bacteriophages infecting multi drug resistant Salmonella serovars isolated from broilers in Egypt.

In this study, we isolated and characterized three phages named as Salmacey1, Salmacey2 and Salmacey3, infecting multi drug resistant Salmonella serovars isolated from broilers in Egypt. The most prevalent Salmonella serovars were S. typhimurium, S. enteritidis, and S. kentucky. All these Salmonella serovars were found to be resistant to more than two of the ten antimicrobial agents tested. Only S. kentucky was found to be resistant to seven antimicrobial agents. Examination of these phage particles by transmission electron microscopy (TEM), demonstrated that two phages (Salmacey1, Salmacey2) were found to belong to family Siphoviridae, and Salmacey3 was assigned to the family Myoviridae. The results of host range assay revealed that these bacteriophages were polyvalent and thus capable of infecting four strains of Salmonella serovars and Citrobacter freundii. Moreover, the two phages (Salmacey1, Salmacey2) had a lytic effect on Enterobacter cloacae and Salmacey3 was able to infect E. coli. All phages could not infect S. para Typhi, Staphylococus aureus and Bacillus cereus. One-step growth curves of bacteriophages revealed that siphovirus phages (Salmacey1, Salmacey2) have burst size (80 and 90pfu per infected cell with latent period 35min and 40min respectively), and for the myovirus Salmacey3 had a burst size 110pfu per infected cell with latent period 60min. Molecular analyses indicated that these phages contained double-stranded DNA genomes. The lytic activity of the phages against the most multidrug resistant serovars S. kentucky as host strain was evaluated. The result showed that these bacteriophages were able to completely stop the growth of S. kentucky in vitro. These results suggest that phages have a high potential for phage application to control Salmonella serovars isolated from broilers in Egypt.

Lysogenic Conversion of the Phytopathogen Ralstonia solanacearum by the P2virus ϕRSY1.

A P2-like phage ϕRSY1 infecting the phytopathogen Ralstonia solanacearum was isolated and characterized. The 40-kb genome of ϕRSY1 showed high sequence similarity to the Ralstonia phage ϕRSA1 and the GMI1000 prophage ϕRSX. The major genomic differences between these phages were the different orientation of the int gene and the gene content close to the cosL. ϕRSY1 and ϕRSX use a 15-base 3' portion of the serine tRNA(GGA) gene as attB, while ϕRSA1 uses a 45-base 3' portion of the arginine tRNA(CCG) gene. The different orientation of int in the genomes means that the gene arrangements in the prophage states are reversed in ϕRSY1 and ϕRSA1. Several putative gene products of ϕRSY1 may affect the bacterium's fitness. ϕRSY1 contains an open reading frame (ORF) that seems to encode a protein similar to Vgr in the type VI secretion system of various bacterial species. ϕRSY1 lysogens showed phenotypic changes including enhanced twitching motility, large colony formation, and easy aggregation of cells, suggesting involvement of this ORF in the changes. In view of these phage gene arrangements, we surveyed prophages in the genomes of various R. solanacearum strains and found that the P2-like phages of R. solanacearum (14 phages) consist of two major groups: the ϕRSY1-type and the ϕRSA1-type. The relationships and evolution of these P2-like phages inferred from our data are discussed in detail.

Dynamic integration and excision of filamentous phage XacF1 in Xanthomonas citri pv. citri, the causative agent of citrus canker disease.

Inovirus XacF1 (7325 nucleotides) is integrated into the genome of Xanthomonas citri pv. citri (Xcc) strains at the host dif site (attB) by the host XerC/D recombination system. The XacF1 attP sequence is located within the coding region of ORF12, a possible phage regulator. After integration, this open reading frame (ORF) is split into two pieces on the host genome. We examined dynamic integration/excision of XacF1 in Xcc strain MAFF 301080 and found that the integration started at 4 h postinfection (p.i.) and peaked at 12 h p.i. Thereafter, the ratio of integrated to free forms remained constant, suggesting equilibrium of integration and excision of XacF1 in the host genome. However, the integrated state became very unstable following a 5'-deletion of ORF12 in XacF1, suggesting that ORF12 plays a key role in the integration cycle of XacF1 in Xcc strains.

Strong antibiotic production is correlated with highly active oxidative metabolism in Streptomyces coelicolor M145.

The Streptomyces genus is well known for its ability to produce bio-active secondary metabolites of great medical interest. However, the metabolic features accompanying these bio-productions remain to be defined. In this study, the comparison of related model strains producing differing levels of actinorhoddin (ACT), showed that S. lividans, a weak producer, had high TriAcylGlycerol (TAG) content indicative of a glycolytic metabolism. In contrast, the strong producer, S. coelicolor, was characterized by low TAG content, active consumption of its polyphosphate (PolyP) stores and extremely high ATP/ADP ratios. This indicated highly active oxidative metabolism that was correlated with induction of ACT biosynthesis. Interestingly, in conditions of phosphate limitation, the ppk mutant had TAG content and ACT production levels intermediary between those of S. lividans and S. coelicolor. This strain was characterized by high ADP levels indicating that Ppk was acting as an Adenosine Di Phosphate Kinase. Its absence resulted in energetic stress that is proposed to trigger an activation of oxidative metabolism to restore its energetic balance. This process, which is correlated with ACT biosynthesis, requires acetylCoA to fuel the Krebs cycle and phosphate for ATP generation by the ATP synthase coupled to the respiratory chain, resulting in low TAG and polyP content of the ACT producing strains.

Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages.

Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All ß- and ß'-subunits were detected in ϕRSF1, but one ß'-subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants.

Two different evolutionary lines of filamentous phages in Ralstonia solanacearum: their effects on bacterial virulence.

The integration and excision of various filamentous phage genomes into and out of their host chromosomes occurs by site-specific recombination. The mechanisms proposed for these events include reactions mediated by phage-encoded recombinases and host recombination systems. Site-specific integration of filamentous phages plays a vital role in a variety of biological functions of the host, such as phase variation of certain pathogenic bacterial virulence factors. The importance of these filamentous phages in bacterial evolution is rapidly increasing with the discovery of new phages that are involved in pathogenicity. Studies of the diversity of two different filamentous phages infecting the phytopathogen Ralstonia solanacearum provide us with novel insights into the dynamics of phage genomes, biological roles of prophages, and the regulation and importance of phage-host interactions.