The effect of suspended particulate material on cyanobacteria—cyanophage interactions in liquid culture

B arnet Y vonne M., D aft , M.J. S tewart , W.D.P. 1984. The effect of suspended particulate material on cyanobacteria-cyanophage interactions in liquid culture. Journal of Applied Bacteriology 56 , 109–115.
The effect of the lytic phage LPP-DUN 1 on the cyanobacterium Plectonema boryanum has been investigated in batch and in continuous cultures in the presence and absence of silt. In batch culture Plectonema without added phage grew normally; the presence of phage caused rapid lysis of the cyanobacterium and the addition of silt prevented lysis by the phage. In continuous culture the numbers of cyanobacterial cells and phage particles oscillated in a reciprocal manner, but the addition of silt damped down the oscillations in Plectonema biomass without decreasing the numbers of phage particles isolated from the cultures. The presence of silt thus appears to protect the cyanobacterium from lysis by phage, although the total numbers of phage particles are relatively unaffected by the silt, at least in the short-term.     

Ethionine-resistant mutants of the filamentous blue-green alga Plectonema boryanum.

Plectonema boryanum mutants that are resistant to ethionine are unable to incorporate ethionine into acid-precipitable material. Ethionine causes bleaching of chlorophyll in sensitive cells.     

Photosynthetic acclimation of the filamentous cyanobacterium, Plectonema boryanum UTEX 485, to temperature and light

Photosynthetic acclimation to temperature and irradiance was studied in the filamentous, non-heterocystous cyanobacterium Plectonema boryanum UTEX 485. Growth rates of this cyanobacterium measured at ambient CO2 were primarily influenced by temperature with minimal effects of irradiance. Both growth temperature and irradiance affected linolenic (18:3) and linoleic acid (18:2) levels in the four major lipid classes in an independent but additive manner. In contrast, photosynthetic acclimation was not due to either growth temperature or irradiance per se, but rather, due to the interaction of these environmental factors. P. boryanum grown at low temperature and moderate irradiance mimicked cells grown at high light. Compared to cells grown at either 29 degrees C/150 micromol m(-2) s(-1) (29/150) or 15/10, P. boryanum grown at either 15/150 or 29/750 exhibited: (1) reduced cellular levels of Chl a and phycobilisomes (PBS), and concomitantly higher content of an orange-red carotenoid, myxoxanthophyll; (2) higher light saturated rates (Pmax) when expressed on a Chl a basis but lower apparent quantum yields of oxygen evolution and (3) enhanced resistance to high light stress. P. boryanum grown at 15/150 regained normal blue-green pigmentation within 16 h after a temperature shift to 29 degrees C at a constant irradiance of 150 micromol m(-2) s(-1). DBMIB and KCN but not DCMU and atrazine partially inhibited the change in myxoxanthophyll/Chl a ratio following the shift from 15 to 29 degrees C. We conclude that P. boryanum responds to either varying growth temperature or varying growth irradiance by adjusting the ability to absorb light through decreasing the cellular contents of Chl a and light-harvesting pigments and screening of excessive light by myxoxanthophyll predominantly localized in the cell wall/cell membrane to protect PSII from over-excitation. The possible role of redox sensing/signalling for photosynthetic acclimation of cyanobacteria to either temperature or irradiance is discussed.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Construction of vector plasmids for the Plectonema boryanum cyanobacterium and creation,on such basis,of the vector-host system

The plasmid composition of the Plectonema boryanum Gom. Cyanobacterium, strain CALU 465, was analyzed. A small pSM1 plasmid, size 1.5, was chosen for genetic engineering. The physical map, constructed for the plasmid, was used to create the pSTS series, i.e. vector construction for the P. boryanum cyanobacterium. A method was selected and tested for the introduction of the vector into host cells. The needed orientation of pSM1 cyanobacterium plasmid within the vector as well as a possible role of its separate elements in inheriting the construction by host were defined.     

Enrichment of the bacteriophage PR4 membrane in phosphatidylglycerol is not essential for phage assembly and infectivity.

The membrane phospholipids of bacteriophage PR4 grown on wild-type Escherichia coli are markedly enriched in phosphatidylglycerol (PG) relative to host phospholipids. To investigate the role of PG in phage assembly and infectivity, we propagated PR4 on an E. coli mutant defective in PG synthesis. The PG content of PR4 grown on the mutant host accounted for 0.4% of the total viral phospholipids, representing a 90-fold decrease in PG relative to the PG content of phage grown on a wild-type host. Phosphatidylethanolamine and phosphatidic acid, the two major phospholipid species present in these phage preparations, accounted for 88.4 and 9.4% of the total viral phospholipids, respectively. This drastic alteration of the phage phospholipid composition had little or no adverse effect on either the stability or infectivity of the phage. We conclude that the enrichment of the PR4 virion in PG does not reflect an absolute structural requirement of the phage and is not essential for phage infectivity.     

A class of ompA mutants of Escherichia coli K12 affected in the interaction of ompA protein and the core region of lipopolysaccharide

Summary A group of ompA mutants of Escherichia coli K12 are described which were sensitive to bacteriophage K3 in a background wild-type for lipopolysaccharide (LPS). With mutant LPS in vivo (lacking some core sugar residues), however, the ompA mutations gave resistance to K3. Outer membrane levels of OmpA protein were normal or near-normal when the mutations resided in either wild-type or mutant LPS backgrounds. Strains in which the mutations occurred in a wild-type LPS background adsorbed K3 phage at the same initial rate and to the same extent as a wild-type strain, but the efficiency of plaquing of the adsorbed K3 was reduced to 25–50% of wild-type levels. Under conditions where a wild-type strain irreversibly adsorbed over 90% of available phage K3 within 3 min, double mutants (ompA mutant, LPS mutant) left 90% of the phage viable after 1h. The 10% of inactivated phage did not form plaques.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1.

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema.

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.     

Organization of nitrogen fixation genes in a nonheterocystous, filamentous cyanobacterium

Abstract The nonheterocystous, filamentous cyanobacterium, Plectonema boryanum fixes nitrogen only under microaerophilic conditions. The organization of nitrogen fixation genes ( nifH, D, K ) in Plectonema was determined by using cloned fragments from the Anabaena nif genes as probes in Southern hybridizations. Regions of Plectonema DNA were homologous to Anabaena nifH, nifD , and nifK genes, and the resulting pattern of hybridization was used to construct a map of nifH, D, K DNA isolated from Plectonema cells grown under non-nitrogen fixing conditions (combined nitrogen and O₂ present). The nifH and nifD genes are on the same 3 kbp Hin dIII fragment, and nifK is on a 1 kbp Hin dIII fragment. All three nif fragments are adjacent to one another on a 12 kbp Cla I fragment.     

Estimation of internal pH in cells of blue-green algae in the dark and under illumination.

Carbonylcyanide m-chlorophenylhydrazone (CCCP) or nigericin induced translocation of H+ In the dark across the cell membrane of blue-green algae Plectonema boryanum and Anacystis nidulans. The direction of the H+ flux depended on the pH of the suspending medium. At acidic pH, an influx of H+ and at alkaline pH an efflux of H+ were observed. It is suggested that the influx takes place at pH'S higher than the "internal" pH and the efflux at pH's lower than that. The internal pH was estimated to be 7.4+/-0.2 for Plectonema boryanum and 7.5+/-0.1 for Anacystis nidulans. Similar H+ changes due to CCP were observed under illumination, where the light induced efflux of H+ was limited by the counter-flux of cations. The internal pH of cells in the light, estimated from the pH-dependent reversion in the rate of the H+ change, was about 8.5.     

Characterization of the genome region encoding an fdxH-type ferredoxin and a new 2[4Fe-4S] ferredoxin from the nonheterocystous, nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110.

A genomic DNA region with four consecutive open reading frames, including an fdxH-type gene, has been sequenced and initially characterized for the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110. The fdxH gene encodes a [2Fe-2S]-type ferredoxin, 98 amino acids in length, with a deduced molecular mass of 10.9 kDa. Conserved residues include two characteristic lysines at positions 10 and 11, shown recently to be important for interaction with nitrogenase reductase (S. Schmitz, B. Schrautermeier, and H. Böhme, Mol. Gen. Genet. 240:455-460, 1993). The gene is transcribed only under anaerobic nitrogenase-inducing conditions, whereas the Plectonema petF gene, encoding a different (type 1) [2Fe-2S] ferredoxin, is only transcribed in cultures growing with combined nitrogen. The fdxH gene was expressed in Escherichia coli as a holoprotein. The purified protein was able to effectively donate electrons to cyanobacterial nitrogenase, whereas PetF from the same organism was not. The occurrence of FdxH in the nonheterocystous genus Plectonema demonstrates for the first time that FdxH-type ferredoxins are not exclusively expressed within heterocysts, as is true for cyanobacteria differentiating these cells for nitrogen fixation under aerobic growth conditions. Two open reading frames that precede fdxH have high similarity to those found at a corresponding location in Anabaena sp. strain PCC 7120. In the latter organism, they are transcribed only under nitrogen-fixing conditions, but the functions of their gene products remain unclear (D. Borthakur, M. Basche, W. J. Buikema, P. B. Borthakur, and R. Haselkorn, Mol. Gen. Genet. 221:227-234, 1990). An fdxB-type gene encoding a 2[4Fe-4S] ferredoxin not previously identified in cyanobacteria is located immediately downstream of fdxH in P. boryanum.     

The nucleotide sequence of the rep gene of cyanobacterial plasmid pSM1

The nucleotide sequence was established for the rep gene of plasmid pSM1 isolated from cyanobacterium Plectonema boryanum CALU 465. Both nucleotide sequence and the encoded amino acid sequences showed 98% homology to the corresponding sequences of small plasmids pPF1, pGL3, pPBS1, pBLX, and pPB1. An active center was identified in the replicative protein sequences.     

Comparative characteristics of physico-chemical and regulating properties of aspartate kinases from three species of cyanobacteria

Aspartokinases have been isolated from the cell-free extracts of Plectonema boryanum, Anabaena variabilis and Synechococcus cedrorum. Their physico-chemical characteristics and peculiarities of retroregulation by amino acids were studied. It has been shown that in P. boryanum cells aspartokinase reaction is catalyzed by only one enzyme, which is similar to the previously described bacterial origin enzymes. Three izoenzymes, which differ in their main properties and amino acids-effectors, have been founded in the cells of A. variabilis. One enzyme with aspartokinase activity, which differs from other cyanobacteria enzymes in some physico-chemical features has been founded in the cells of S. cedrorum.     

INFLUENCE OF IRON LIMITATION AND NITROGEN SOURCE ON GROWTH AND SIDEROPHORE PRODUCTION BY CYANOBACTERIA1

To characterize the mobilization and uptake of iron by cyanobacteria, 14 species were screened for ability to scavenge iron in a competitive system. The cyanobacteria exhibited a range of growth responses to iron limitation which could be separated into three groups, and a representative species from each group was chosen for further study. Effects of iron-limitation on growth and siderophore production of Anacystis nidulans R2, Anabaena variabilis ATCC 29413, and Plectonema boryanum UTEX 581 were determined. Both A. nidulans R2 and A. variabilis showed a reduced rate of growth with decreased available iron concentration (PFe 17–19). Growth rates increased with further reduction in the level of available iron (pFe 20 to pFe 21). The increase in growth rate occurred at the same available iron concentration as the initiation of extracellular siderophore production. In contrast, the growth of P. boryanum decreased with decreasing available iron levels. No siderophore production was detected from P. boryanum cultures. The growth kinetics of siderophore-producing species differ from traditional nutrient-limited growth kinetics and clearly reflect the presence of a high affinity, siderophore-mediated iron transport system in A. nidulans R2 and A. variabilis. Iron-limited growth kinetics more similar to traditional nutrient-limited growth kinetics were found in P. boryanum. The available nitrogen source influenced amount of siderophore produced and concentration of available iron which induced siderophore production. Siderophores were produced at high iron concentrations (pFe 18) when A. variablilis cultures were grown in the absence of combined nitrogen source. When nitrate was supplied to the culture, iron concentrations had to be reduced to pFe 20 before siderophores were produced. Cells grown on nitrogen also produced greater than two times the amount of siderophore compared with nitrate grown cells. This may be indicative of an increased demand for iron by nitrogen fixing A. variabilis Cultures.     

The Oxygen-Scavenging System Protecting Nitrogenase from Oxygen in Cells of Blue-Green Algae

There is a heat stable oxygen-scavenging system (OSS) associated with membrane which reduces oxygen endogenously in cells of blue-green algae. Addition of the OSS to cell suspension of heterocystous oxygen sensitive Anabaena mutant and non-heterocystous Pleetonema boryanum led to an increase in their nitrogenase activity by 10–100-fold higher than those under microaerobic condition and also could restore effectively their acetylene reduction activity at higher oxygen concentration since the oxygen presented was reduced effectively. The results suggest that the OSS possesses a function protecting nitrogenase from oxygen in cells. Furthermore, it was found that the efficiency of reducing oxygen of OSS from the Anabaena mutant and Plectonema was lower than those from Anabaena wild and Gloeocapsa in atm. oxygen level. This may be ralated with the susceptibility of nitrogen fixation to oxygen in the cells of Anabaena mutant and Plectonema. The present study firstly indicades the relationship between the heat stable OSS associated with membrane and the mechanism of protecting nitrogenase from oxygen in cells of blue-green algae. Activities of catalase, peroxidase and superoxide dismutase do not show obvious difference in cellfree extract of Anabaena wild and mutant. Methyl viologen can induce nitrogenase activity of Anabaena mutant by subverting a portion of electon flow to accelerate oxygen reduction.     

INTERACTION OF PLECTONEMA BORYANUM (CYANOPHYCEAE) AND THE LPP-CYANOPHAGES IN CONTINUOUS CULTURE1

Continuous culture techniques are used to study long-term population interactions between Plectonema boryanum Gomont, a filamentous bluegreen alga, and the LPP-viruses which infect it. After LPP-I (virulent cyanophage) infection of sensitive algae, 3 oscillations occur in cell density with concomitant oscillations in virus titer before final stabilization of both algal and viral concentrations. After LPP-ID and LPP-2 (temperate viruses) infection, oscillation in cell density occurred with burst of virus particles. Resistant algae always repopulated the chemostat; lysogeny was not established. The interaction between Plectonema that was resistant to virus infection and the 3 LPP-cyanophages resulted in rapid elimination of the viruses from the chemostat in the effluent. When lysogenic P. boryanum was tested, a law population of virus was present in the chemostat throughout the incubation period indicative of spontancous induction. Clones of lysogenic algae were isolated.     

The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10⁴ and 10⁶ pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.     

Host re-identification of cyanophage PP and its implications for host range and specificity

Dear Editor, In this study,we re-identified the hosts of cyanophage PP.Twenty-three candidate algal strains were tested,and the results indicated that seven strains belonging to Plectonema and Phormidium are the hosts of cyanophage PP,including two previously reported filamentous cyanobacteria,Plectonema boryanum and Phormidium foveolarum (Zhao et al.2002).However,several species or strains within the two genera were found not to be hosts of cyanophage PP,implying that the host range is relatively specific.     

Uracil-DNA glycosylase causes 5-bromodeoxyuridine photosensitization in Escherichia coli K-12.

An Escherichia coli uracil-DNA glycosylase-defective mutant (ung-1 thyA) was more resistant than its wild-type counterpart (ung+ thyA) to the killing effect of UV light when cultured in medium containing 5-bromouracil or 5-bromo-2'-deoxyuridine (BrdUrd). The phenotype of resistance to BrdUrd photosensitization and the uracil-DNA glycosylase deficiency appeared to be 100% cotransduced by P1 phage. During growth with BrdUrd, both strains exhibited similar growth rates and 5-bromouracil incorporation into DNA. The resistant phenotype of the ung-1 mutant was observed primarily during the stationary phase. In cells carrying 5-bromouracil-substituted DNA, mutations causing resistance to rifampin and valine were induced by UV irradiation at a higher frequency in the wild type than in the ung-1 mutant. This Ung-dependent UV mutagenesis required UmuC function. These results suggest that the action of the uracil-DNA glycosylase on UV-irradiated 5-bromouracil-substituted DNA produces lethal and mutagenic lesions. The BrdUrd photosensitization-resistant phenotype allowed us to develop a new, efficient method for enriching and screening ung mutants.