Morphology and General Characteristics of Bacteriophages Infectious to Robinia pseudoacacia Mesorhizobia

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley’s classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10–25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100–120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 μg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from ~39 kb for group C phages to ~80 kb for B2.     

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p _E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p _E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998     

Roles of Exopolysaccharides and Lipopolysaccharides in the Adsorption of the Siphovirus Phage NM8 to Rhizobium meliloti M11S Cells

The exopolysaccharides produced by Rhizobium meliloti M11S inhibited nonspecifically the adsorption of phage NM8 by coating the cells. But lipopolysaccharides (LPS) had a specific inhibitory effect. Only the polysaccharide moiety of LPS, composed of glucose, glucosamine, galactose, 3-deoxy-D-manno-octulosonic acid (KDO), and large amounts of sialic acid, inhibited phage adsorption; neither the lipid A moiety nor a cellular glucan was involved. Rhizobium strains lacking sialic acids did not bind phage NM8. Inhibition of phage binding by lectin specific for N-acetylneuraminic acid demonstrated that phage NM8 bound to sialic acids. Preincubation of the phage with monosaccharides showed that inactivation of phage was very stereospecific for N-acetylneuraminic acid. Phage adsorption was also strongly inhibited by N-acetylglucosamine, which is not present in the LPS. Therefore, the receptor for phage NM8 appears to be a saccharide site, probably involving the acetyl groups of sialic acids. Received: 8 March 1996 / Accepted: 29 June 1996     

Morphology and General Characteristics of Lytic Phages Infective on Strains of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Biological characteristics of three isolated phages (SR1, SR2, and SR3) lytic against three Bradyrhizobium japonicum strains were studied. These phages had no cross-infectivity among the host strains. Phage morphology indicates that they belonged to Siphoviridae (long noncontractile tail; SR1 and SR2) and Podoviridae (short tail; SR3) classes of bacteriophages. Lytic cycle of phages studied under identical conditions showed a distinct adsorption rate (67.3–99.1%), latent period (150–300 min), rise period (60–150 min), and burst size (110–200 pfu/cell). Stability in liquids and inactivation by osmotic shock, thermal, and ultraviolet irradiation were also distinct in this heterogeneous phage group. Influence of soil factors such as temperature, soil moisture, soil pH, and degree of phage adsorption to the soil on phage survival was determined. Major percent of free infective phages were obtained after desorption of phages from soil. Overall, temperature appeared to be the most important parameter affecting rhizobiophage survival in the soil.     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Isolation and characterization of a lipopolysaccharide-specific bacteriophage of Pseudomonas aeruginosa

Phage H22 was isolated from sewage using Pseudomonas aeruginosa NCTC 8505 (serotype 0:3) as the host. Although not O-specific, this phage was found to have lipopolysaccharide (LPS) as a receptor. The broad host-range and lack of O-specificity of the phage suggested that its receptor site was in the core region of the LPS. Phage H22 had a Bradley type A structure. It was unaffected by chloroform and diethyl ether, and was stable between pH 5 and 8 and in the temperature range 0 to 60 degrees C. The adsorption rate constant was 14.6 X 10(-9) ml min-1. The phage had a latent period of 43 min, with a rise time of 18 min and a burst size of 6. The adsorption of phage to whole cells and LPS occurred over a broad pH range. Maximum adsorption occurred at 50 degrees C and pH 7.5 in the presence of 0.001 M Ca2+.     

Beyond the A‐layer: adsorption of lipopolysaccharides and characterization of bacteriophage‐insensitive mutants of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a fish pathogen that causes furunculosis. Antibiotherapy used to treat furunculosis in fish has led to resistance. Virulent phages are increasingly seen as alternatives or complementary treatments against furunculosis in aquaculture environments. For phage therapy to be successful, it is essential to study the natural mechanisms of phage resistance in A. salmonicida subsp. salmonicida. Here, we generated bacteriophage‐insensitive mutants (BIMs) of A. salmonicida subsp. salmonicida, using a myophage with broad host range and characterized them. Phage plaques were different depending on whether the A‐layer surface array protein was expressed or not. The genome analysis of the BIMs helped to identify mutations in genes involved in the biogenesis of lipopolysaccharides (LPS) and on an uncharacterized gene (ASA_1998). The characterization of the LPS profile and gene complementation assays identified LPS as a phage receptor and confirmed the involvement of the uncharacterized protein ASA_1998 in phage infection. In addition, we confirmed that the presence of an A‐layer at the bacterial surface could act as protection against phages. This study brings new elements into our understanding of the phage adsorption to A. salmonicida subsp. salmonicida cells.     

BVPaP-3, a T7-Like Lytic Phage of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Its Isolation and Characterisation

The increasing emergence of antibiotic-resistant bacteria has produced a growing interest among scientists in bacteriophages as alternative antimicrobial agents. This article reports a lytic phage against an antibiotic-resistant strain of Pseudomonas aeruginosa. Phage BVPaP-3 is a member of the Podoviridae family and morphologically similar to the T7-like phage gh-1. The phage has a hexagonal head of 58–59 nm in diameter and a short tail of 10 × 8 nm. It is stable at a wide range of pH (6–10) and temperatures (4–40°C). Its optimal growth temperature is 37°C and the adsorption rate constant is 1.19 × 10⁻⁹. Latent and eclipse periods are 20 and 15 min, respectively, and the burst size is 44 after 35 min at 37°C. The phage has a DNA size of 41.31 kb and a proteome of 11 proteins. The major protein is 33 kDa in size.     

The requirement for exopolysaccharide precedes the requirement for flavolan-binding polysaccharide in nodulation of Leucaena leucocephala by Rhizobium loti

Rhizobium loti strain PN4115 (NZP2213 str-1) ineffectively nodulates Leucaena leucocephala, i.e., strain PN4115 induces nodulation (Nod⁺) and is able to invade these nodules (Inv⁺), but fails to fix nitrogen (Fix^–). Strain PN4115 does not synthesize a flavolan-binding polysaccharide (FBP), which is synthesized by the fully effective (Nod⁺Inv⁺Fix⁺) R. loti strain PN184 (NZP2037 str-1). The FBP may offer protection from prodelphinidin-rich flavolans synthesized by Lc. leucocephala. In this work, we show that exopolysaccharide (EPS)-negative mutants derived from strain PN4115 have a more severe ineffective phenotype (Nod⁺Inv^–Fix^–) on Lc. leucocephala than strain PN4115. This suggests that EPS from strain PN4115 is functional during invasion of Lc. leucocephala and that the requirement for EPS precedes the requirement for FBP. Received: 8 October 1996 / Accepted: 11 December 1996     

Identification of distinct lipopolysaccharide patterns among <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> and <Emphasis Type="Italic">Y. enterocolitica</Emphasis>-like bacteria

The lipopolysaccharide (LPS) of strains representing various serotypes of Yersinia enterocolitica and Y. enterocolitica-like bacteria was studied by deoxycholate-PAGE and silver staining analysis. Four main types of LPS were detected based on the O-polysaccharide (O-PS): (i) LPS with homopolymeric O-PS, (ii) LPS with ladder-forming heteropolymeric O-PS, (iii) LPS with single-length O-PS, and (iv) semi-rough LPS without O-PS. Within the first three types, several subvariants were detected. Selected serotypes representing all above LPS types are sensitive to bacteriophage ϕR1-37 indicating that they share the phage receptor, a hexasaccharide called outer core in Y. enterocolitica O:3. Whereas phage ϕR1-37-resistant mutants of homopolymeric O-PS have lost only the outer core, those of ladder-forming or single-length O-PS have lost also the O-PS suggesting that in the latter ones the outer core is bridging between O-PS and lipid A-core. This work forms a basis of further structural, biochemical and genetic studies of these LPSs.     

Osmotic water permeability of isolated vacuoles

We measured the osmotic water permeability (P _os) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, P _os values were in the range 200–1000 μm s⁻¹. The change in membrane surface area induced by an osmotic gradient was smaller than 2–6%. The vacuolar P _os values for red beet and onion were reduced by 1 mM HgCl₂, to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5 mM β-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl₂-treated onion vacuoles, the reduced P _os (56 μm s⁻¹) had a low activation energy (approx. 6 kJ mol⁻¹), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low. Received: 18 February 1999 / Accepted: 21 June 1999     

Mycoflora and mycotoxins natural occurrence in corn from entre rios province, Argentina

Corn samples were collected in 1999 from three departments of Entre Réos province, Argentina, and were surveyed for mould contamination and natural occurrence ofFusarium mycotoxins, ochratoxin A and aflatoxins.Fusarium verticillioides was the most prevalent fungal species recorded at all departments. Zearalenone, deoxynivalenol and ochratoxin A were not found in any samples. Only one of the 52 corn samples analysed was contaminated with aflatoxin B₁ (17 μg/kg). Fumonisin B₁ was found in 58 % of samples (range of positive samples: 47– 3,347 μg/kg), fumonisin B₂ in 33.0 % (range of positive samples: 23–537 μg/kg) and fumonisin B₃ in 25.0 % (range of positive samples: 24–287 μg/kg) of them. This is the first report on the natural occurrence of mycotoxins in corn from Entre Ríos province, Argentina. Levels of fumonisins were lower than detected in other Argentinian provinces.     

In Vitro Management of Hospital <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Biofilm Using Indigenous T7-Like Lytic Phage

Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1–HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.     

Phage Therapy of Coral White Plague Disease: Properties of Phage BA3

The bacteriophage BA3 multiplies in and lyses the coral pathogen Thalassomonas loyana. The complete genome of phage BA3 was sequenced; it contains 47 open reading frames with a 40.9% G + C content. Phage BA3 adsorbed to its starved host in seawater with a k = 1.0 × 10⁻⁶ phage ml⁻¹ min⁻¹. Phage therapy of coral disease in aquarium experiments was successful when the phage was added at the same time as the pathogen or 1 day later, but failed to protect the coral when added 2 days after bacterial infection. When the phages were added 1 day after coral infection, the phage titer increased about 100-fold and remained present in the aquarium water throughout the 37-day experiment. At the end of the experiment, the concentration of phages associated with the corals was 2.5 ± 0.5 × 10⁴ per cm² of coral surface. Corals that were infected with the pathogen and treated with phage did not transmit the disease to healthy corals.     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999     

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Received: 16 March 1999 / Accepted: 16 April 1999     

Independent production of two molecular forms of a recombinant Rhizopus oryzae lipase by KEX2-engineered strains of Saccharomyces cerevisiae

A mixture of rProROL having the full-length prosequence (97 amino acids) for a recombinant lipase of Rhizopus oryzae (rROL) and r28ROL having 28 amino acids of the same prosequence has been produced as active forms by Saccharomyces cerevisiae [Takahashi et al. (1998) J Ferment Bioeng 86: 164–168]. However, the separation of rProROL and r28ROL has not been successful due to their identical behavior on column chromatographs, presumably because of the similarity of their surface properties. The independent production of two different molecular forms of rROL was carried out using KEX2-engineered strains of S. cerevisiae, since r28ROL was predicted to be a product from rProROL by a Kex2-like protease. rProROL was successfully obtained by expression of the ROL gene in the S. cerevisiae kex2 strain in which the KEX2 gene encoding Kex2p was disrupted, while r28ROL was obtained by co-expression of the gene (KEX2Δ613) encoding the soluble form of the C-terminal truncated Kex2 protease (sKex2p). The specific lipase activities of rProROL and r28ROL were 92.9 U/mg and 140 U/mg, respectively. rProROL was stable at pH 2.2–8.0, and showed the optimal reaction temperature to be 30–35 °C with a T ₅₀ of 55 °C (T ₅₀ is the temperature resulting in 50% loss of activity). The values for r28ROL were pH 3.0–10.0, 25–30 °C, and 40 °C, respectively. rProROL was an N-linked glycosylated form, but r28ROL was not. The enhanced thermostability of rProROL did not seem to be due to the N-linked glycosylation, as judged by the results of the Endo H treatment. rProROL had the highest esterase activity toward p-nitrophenyl laurate (C₁₂), whereas r28ROL had the highest esterase activity toward p-nitrophenyl caprylate (C₈) and stearate (C₁₈). These results suggest that the distinct properties of these two forms of lipase are caused by the different length of the ROL prosequence. Received: 26 January 1999 / Received last revision: 24 May 1999 / Accepted: 4 June 1999     

Phage abortive infection of Bacillus licheniformis ATCC 9800; identification of the abiBL11 gene and localisation and sequencing of its promoter region

The virulent bacteriophage BL11 infects almost all Bacillus licheniformis strains tested, including the industrial bacitracin-producing B. licheniformis 19. B. licheniformis ATCC 9800, however, was virtually insensitive to phage BL11 infection, and all of the few surviving progeny phages proved to be mutants. The phage-resistance mechanism was neither inhibition of adsorption, nor restriction or exclusion provided by a resident prophage, but was, instead, of another type. Phage BL11 adsorbed well on to ATCC 9800 cells, its DNA was injected, but replication of phage DNA was inhibited and the infected cells died. Thus, the mechanism of phage resistance was identified as abortive infection (AbiBL11). The so-called abiBL11 gene was identified on the chromosome of strain ATCC 9800 by Tn917PF1 transposon mutagenesis. Part of the abiBL11 gene from the phage-sensitive ATCC 9800::Tn917PFI was cloned. Gene-disruption analysis, based on Campbell-type integration, showed that a 0.3-kb EcoRI fragment contained the 5′ end of abiBL11. The promoter region of abiBL11 was identified using promoter- and terminator-probe plasmids. The deduced sequence (206 amino acids) of the N-terminal part of abiBL11 showed no significant homology to known abortive-infection genes, but did show homology to a Saccharomycescerevisiae gene coding for a serine/threonine protein kinase (RCK1). Received: 1 April 1999 / Received revision: 12 July 1999 / Accepted: 13 July 1999     

φ20, A Temperate Bacteriophage Isolated from Bacillus anthracis Exists as a Plasmidial Prophage

This study describes the isolation of temperate B. anthracis phages, from 4 out of 20 B. anthracis strains screened, by use of the inducing agents mitomycin C and UV light. Phage φ20 isolated from B. anthracis Sterne 34F₂ (pXO1⁺ pXO2⁻) was shown to have double-stranded DNA of size 48756 bp and a restriction site map showing nine sites for enzymes BamHI, BglII, and SstI is included. The φ20 genome was found to exist as a plasmidial prophage and the phage itself to have a polyhedral head of diameter 65 nm and tail 217 nm long and 15 nm wide.     

Transduction inMycobacterium phlei

Transduction, the transfer of genetic information from one bacterial host to another by bacteriophage, has been demonstrated inMycobacterium phlei. The ability to utilized-xylose as a sole carbon source could be transferred fromM. phlei strain SN109 (xyl ⁺,str-s) toM. phlei strain F89 (xyl ⁻,str-r) by a bacterially sterile, DNase-treated phage Bo2-lysate of donorM. phlei SN109. The marker was expressed within 15 minutes of phage infection, but the recipient bacteria had to be in early stationary phase for transduction to occur. Controls employed in these experiments were bacteria incubated with heat-killed phage, with phage plus antiphage serum, or with sterile broth in place of phage. Phage conversion, the appearance of a new character due solely to phage infection, could not account for the observed phenomenon since phage Bo2, which was not previously propagated on axyl ⁺ donor, failed to confer the ability to utilize xylose on axyl ⁻ repicient. High frequency transduction (HFT) also could be demonstrated by using phage lysates obtained fromxyl ⁺ transductants. This work was presented in part at the 71st Annual Meeting of the American Society for Microbiology, Minneapolis, 2–8 May, 1971. Submitted by the senior author in partial fulfillment of the requirement for the Ph.D. degree at the Department of Microbiology, Loyola University — Stritch School of Medicine, Maywood, Illinois.