Lytic Enzymes toward Aniline-assimilating Rhodococcus erythro-polis AN-13: Purification and Characterization

Three lytic enzymes, C-2, C-4 and C-5, capable of lysing cells of Rhodococcus erythropolis AN-13 were purified from the cultural filtrate of Flavobacterium species SH-548 by (NH₄)₂S0₄ fractionation and column chromatographies on CM-Toyopearl and SP-Sephadex. The three purified enzymes gave single protein bands on polyacrylamide gels. C-4 and C-5 were stable between pH 3.0 and 12.5, and C-2 between pH 5.5 and 11.0. The molecular weights of C-4 and C-5 were 26,000 and that of C-2 was 36,000, as judged on sodium dodecylsulfate-polyacrylamide gel electrophoresis. C-4 and C-5 also showed proteolytic activity toward casein, but C-2 did not exhibit such activity. C-2 showed higher specific lytic activity toward cells of R. erythropolis AN-13 than C-4 and C-5.     

Role of Predatory Bacteria in the Termination of a Cyanobacterial Bloom

Changes in cyanobacterial abundance and in the occurrence of bacteria of bacteria capable of lysing cyanobacteria were monitored over a period of 6 months (May to October 1998) in eutrophic Brome Lake (Quebec, Canada), in which dense cyanobacterial blooms recur regularly. By screening lake water, we isolated two strains of lytic bacteria, from the family Cytophagaceae. When tested on 12 cyanobacteria and 6 heterotrophic bacteria, strain 1 lysed only Anabaena flos-aquae and strain 2 lysed only Synechococcus cedorum, Synechococcus leopoliensis, Synechococcus elongatus, and Anacystic nidulans: both liquid and agar-grown cultures of these cyanobacteria were lysed. The number of plaque forming units of bacteria increased dramatically during the decline of the bloom. The results are consistent with an important role for these host-specific lytic bacteria in control and elimination of cyanobacterial blooms in this lake.     

Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry

Aims: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. Methods and Results: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. Conclusions: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage‐related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. Significance and Impact of the Study: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.     

Phage susceptibility and plasmid profile analysis of Sinorhizobium fredii

This is the first report identifying bacteriophages and documenting megaplasmids of Sinorhizobium fredii. Plasmid DNA content and bacteriophage typing of eighteen strains of S. fredii were determined. S. fredii strains fell into ten plasmid profile groups containing 1 to 6 plasmids, some evidently larger than 1000 MDa. Twenty-three S. fredii lytic phages were isolated from soil, and they lysed six different S. fredii strains. The host range and plaque morphology of these phages were studied. Susceptibility to S. fredii phages was examined for S. meliloti; Rhizobium leguminosarum bvs. viceae, trifolii and Phaseoli; R. loti; Bradyrhizobium japonicum; B. elkanii and Bradyrhizobium sp. (Arachis). Several phages that originally lysed S. fredii strain USDA 206 also lysed strains of all three S. fredii serogroups described originally by Sadowsky et al. Phages that infected S. fredii strains USDA 191 and USDA 257 were highly specific and lysed only serogroup 193 strains. S. meliloti strains L5-30 and USDA 1005 were lysed by three of the phages that lysed S. fredii strain USDA 217. No other Rhizobium or Bradyrhizobium strain tested was susceptible to lysis by any of the S. fredii phages. The present investigation indicates that phage susceptibility in conjunction with plasmid profile analysis may provide a rapid method for identification and characterization of strains of S. fredii.     

Gas Chromatographic Analysis of Volatile Esters in Wines

We isolated a new aerobic gram negative collagenolytic bacterium, strain CRZV₁. This strain is yellow pigmented, non motile; it poorly degrades the sugars and its proteolytic activity corresponds to one enzyme, a collagenase which is produced in media with collagen, or collagen like substrates. The strain CRZV_l, presents many similarities with Flavobacteria. However, the GC content of the DNA, which is 65%, excludes our collagenolytic strain from the genus Flavobacterium, where all the species have GC content between 30 and 42%. All the strains, already described as Flavobacteria, the GC contents of which are situated between 55 and 70%, are actually unclassified. They could be included in the genus Empedobacter. This genus is not in the approved list of bacterial names, so we only consider this new collagenolytic bacteria as a gram negative, aerobic, yellow pigmented bacteria.     

Identification of a flavobacterium strain virulent against Giardia lamblia cysts

We have isolated from a Kentucky stream a bacterial strain capable of killing the cyst form of Giardia lamblia. This bacterium, designated Sun4, is a Gram-negative, aerobic rod which produces a yellow pigment, but not of the flexirubin-type. Although true gliding motility has not been observed in Sun4, this strain does exhibit a spreading colony morphology when grown on R2A agar. Strain Sun4 has been identified by 16S rRNA sequencing and phylogenetic analysis as belonging to the genus Flavobacterium, and is most closely related to Cytophaga sp. strain Type 0092 and associated Flavobacterium columnare strains. Lipid analysis also identified fatty acids characteristic of the Cytophaga–Flavobacterium group of bacteria. In culture, Sun4 is able to degrade casein and cellulose, but not chitin, gelatin, starch, or agar. Degradation of Giardia cysts by Sun4 appears to require direct cellular contact as neither cell-free extracts nor cells separated from the cysts by dialysis membranes showed any activity against cysts. Activity against Giardia cysts is rapid, with Sun4 killing over 90% of cysts within 48 h. Strain Sun4 requires elevated levels of Ca²⁺ for optimal growth and degradative activity against Giardia cysts. We propose that bacterial strains such as Sun4 could be used as biological control agents against Giardia cysts in drinking water treatment systems.     

Purification and Properties of Phage HM 7-Induced Lytic Enzyme of Clostridium saccharoperbutylacetonicum

A lytic enzyme was isolated from phage HM 7-induced lysate of Clostridium saccharoperbutylacetonicum, and purified about 200-fold by precipitation with ammonium sulfate, gel filtration with Sephadex G–75 and ampholine isoelectric focusing. The purified lytic enzyme had an apparent homogeneity on disc-electrophoresis, and the character of acidic protein showing isoelectric point at pH 4.0. The molecular weight of lytic enzyme was estimated to be about 100,000 from the result of SDS-polyacrylamide gel electrophoresis. The optimum pH for the lytic enzyme activity was 6.5. Maximum activity occurred at 30 to 35°C, and at the ionic strength of 0.04 m or above. The lytic enzyme activity was stimulated about 140% by 10?³ m EDTA. The lytic enzyme lysed the living cells, but it had a narrow specificity which was restricted to a certain species of Clostridium such as Cl. saccharoperbutylacetonicum, Cl. butyricum, Cl. botulinum, Cl. sporogenes, and Cl. thiaminolyticum.     

A comparison of strains of King's group IIb of Flavobacterium with Flavobacterium meningosepticum

Twenty-two strains of Flavobacterium recently isolated from patients and other sources were compared with 6 strains of Flavobacterium meningosepticum and 3 strains of King's Flavobacterium group IIb. The field strains were found to resemble group IIb in their characteristics.All 31 strains of Flavobacterium gave similar results in 28 phenotypic tests; the DNA base compositions of 18 phenotypically representative strains ranged from 35 to 39% GC. Within this group, the 6 strains of F. meningosepticum were phenotypically homogeneous, had a % GC of 36.9, and differed consistently from the 25 strains of group IIb only in the pale colour of their pigment, slowness of pigment production, and inability to hydrolyse starch. All 25 strains of group IIb differed in at least 6 tests from the 6 strains of F. meningosepticum, although not the same 6 tests in each case. Antisera to F. meningosepticum agglutinated 10 strains of group IIb.     

Bacteriocinogenesis in cells of Xenorhabdus nematophilus and Photorhabdus luminescens: Enterobacteriaceae associated with entomopathogenic nematodes

Summary— Xenorhabdus nematophilus FI strain and Photorhabdus luminescens NC19 strain produced bacteriocins after mitomycin C treatment and under natural conditions respectively. The ultrastructure of these two strains was described and compared to the ultrastructure of untreated or normal cells. After image processing of purified bacteriocins we found morphological homology in infected cells with protoplasmic rods in longitudinal section and hexagonal aggregates in transversal section. We concluded that these particular structures, so-called ‘lattice structures’ and previously interpreted as ‘photosomes’, are in fact the early stages of in situ production of bacteriocins in these two bacterial genera. Natural occurrence of Photorhabdus spp bacteriocinogenesis was observed in other strains, while other lysogenic strains of Xenorhabdus spp are lysed after a mitomycin C treatment.     

Specificity patterns of Agrobacterium tumefaciens phages

Summary Lysogeny was not detected in 10 strains of A. tumefaciens by plating techniques or ultra-violet induction. Fifteen phages were isolated from raw sewage against 13 cultures of A. tumefaciens and purified by single-plaque selections. No phage lysed all of the strains of A. tumefaciens tested; one phage lysed only a single strain; 2 other phages attacked 7 strains. Ten of the 15 phages lysed no more than 3 strains. Three host strains showed identical phage susceptibilities. No relationship was noted between susceptibility to phage and ability of a strain to incite crown galls.Thirteen phages lysed at least 1 of 4 strains of A. radiobacter, but none attacked single strains of A. rubi or A. pseudotsugae. Eleven phages lysed the one strain of A. rhizogenes used. None of the phages had identical host ranges with respect to all the Agrobacterium spp. tested. Similarly none of 5 selected phages attacked any one of 59 strains of bacteria from 12 different genera including 35 strains of rhizobia. Within the limits of this study the phages used were genus-specific.Published with approval of the Director, Wisconsin Agricultural Experiment Station, Madison, Wisconsin, U.S.A. 53706.     

Diversity of cultivable bacteria isolated from the water column and bottom sediments of the Kara Sea shelf

In this work, the results of microbiological and molecular genetic investigation of the microorganisms inhabiting the Kara Sea and the adjacent Yenisei and Gydanskii Bays are presented. The microorganisms isolated from the samples collected in the studied area belonged to 4 phyla and 11 genera. Bacteria of two phyla, Firmicutes and Actinobacteria, prevailed; representatives of the Gammaproteobacteria and Bacteroidetes were isolated as well. According to their phenotypic properties, the obtained pure cultures were classified with the genera Streptomyces, Rhodococcus, Micrococcus, Bacillus, Pseudomonas, Acinetobacter, Flavobacterium, and Marinococcus. Analysis of the obtained nucleotide sequences of the 16S rRNA genes confirmed that the isolates belonged to the genus Bacillus. One strain was reidentified as Brevibacillus laterosporus, and two strains were identified Aeromonas piscicola and Plantibacter sp. The results of the study of the enzymatic activity of the obtained pure psychrotolerant cultures suggest that the microbial community is actively involved in the destruction processes occurring in the studied area.     

Isolation of lytic bacteriophage against Vibrio harveyi

Aims: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Methods and Results: Water samples from discharge channels and grow‐out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage‐enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Conclusions: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Significance and Impact of the Study: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.     

伊犁河流域春季可培养细菌多样性及其胞外酶检测

利用可培养法对新疆伊犁河流域水体和沉积物中细菌多样性进行分析，以期初步阐明流域河流可培养细菌群落结构。采用5种琼脂培养基分离纯化可培养细菌，依据其16S rRNA基因序列进行系统发育分析，并运用平板法对纯化菌株的胞外酶产生情况进行检测。序列分析结果表明，225株细菌分别属于变形菌门γ亚群(Gamma-pseudomonadota, 56.44%)、放线菌门(Actinomycetota, 18.22%)、厚壁菌门(Firmicutes, 14.22%)、变形菌门α亚群(Alpha-pseudomonadota, 4.89%)、变形菌门β亚群(Beta-pseudomonadota, 4%)、拟杆菌门(Bacteroidota, 0.44%)和异常球菌-栖热菌门(Deinococcota, 0.44%)等7个大的系统发育类群，41个属84个种。其中假单胞菌属(Pseudomonas,42.22%)、不动杆菌属(Acinetobacter,9.33%)和芽胞杆菌属(Bacillus,9.33%)为优势菌属。菌种分布结果显示，伊犁河流域主要支流和干流中可培养细菌地域分布性强。分离菌株产胞外酶...     

Biosurfactants,an help in the biodegradation of hexadecane? The case of <Emphasis Type="Italic">Rhodococcus</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis> strains

The roles of the extracellular biosurfactants produced by two bacterial strains, Pseudomonas aeruginosa GL1 and Rhodococcus equi Ou2, in hexadecane uptake and biodegradation were compared. For this purpose, cell hydrophobicity and production of glycolipidic biosurfactants were evaluated during bacterial growth on hexadecane, as well the effects of these biosurfactants on culture supernatants properties i.e., surface and interfacial tensions, and emulsification and pseudosolubilization capacities. The results showed that the role of biosurfactants was different in these two strains and was directly related to the hydrophobicity of the bacterial cells concerned. Extracellular biosurfactants produced by strain R. equi Ou2 had only a minor role in hexadecane degradation. Direct interfacial accession appeared to be the main mechanism for hexadecane uptake by the hydrophobic cells of strain R. equi Ou2. On the contrary, the biosurfactants produced by P. aeruginosa GL1 were required for growth on hexadecane, and their pseudosolubilization capacity rather than their emulsification capacity was involved in substrate degradation, allowing uptake from hexadecane micelles by the hydrophilic cells of this bacterium. The roles of biosurfactants thus differ widely among bacteria degrading hydrophobic compounds. J.-P. Vandecasteele—in retirement     

The <Emphasis Type="Italic">cadA</Emphasis> Gene in Cadmium-Resistant Bacteria from Cadmium-Polluted Soil in the Zhangshi Area of Northeast China

Cadmium-resistant bacteria were isolated from the farmland soil in Zhangshi Irrigation Area in Shenyang of Northeast China, an area has been polluted by heavy metals, especially cadmium, for more than 40 years. The cadA gene was detected in 4 Bacillus strains and for the first time in one Flavobacterium strain. The high sequence identity (93%–99%) of cadA gene, shared indels in different bacterial species and genera, and the phylogenetic incongruence between 16S rDNA gene tree and cadA gene tree suggested that lateral gene transfer (LGT) occurred among Bacillus and Flavobacterium spp. The LGT of cadA gene might play a vital role in promoting the spread of cadmium-resistant phenotypes throughout soil microbial communities. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.     

Lysis of Pediococcus and Brevibacterium Cells by Cell-Wall Lytic Enzymes,L3, L11, ALE and Lysozyme

Several species belonging to the genera Pediococcus and Brevibacterium, which have resistant cell walls against the usual cell-disrupting methods, were effectively attacked by new cell-wall lytic enzymes, L₃, L₁₁ and ALE which were obtained from a Streptomyces sp., a Flavobacterium sp., and a Staphylococcus sp., respectively. Among them, the L₃ enzyme was mostly effective to all pediococcal and brevibacterial strains.     

Biodegradation of polyethylene glycol by symbiotic mixed culture (obligate mutualism)

Neither Flavobacterium sp. nor Pseudomonas sp. grew on a polyethylene glycol (PEG) 6000 medium containing the culture filtrate of their mixed culture on PEG 6000. The two bacteria did not grow with a dialysis culture on a PEG 6000 medium. Flavobacterium sp. grew well on a dialysis culture containing a tetraethylene glycol medium supplemented with a small amount of PEG 6000 as an inducer, while poor growth of Pseudomonas sp. was observed. Three enzymes involved in the metabolism of PEG, PEG dehydrogenase, PEG-aldehyde dehydrogenase and PEG-carboxylate dehydrogenase (ether-cleaving) were present in the cells of Flavobacterium sp. The first two enzymes were not found in the cells of Pseudomonas sp. PEG 6000 was degraded neither by intact cells of Flavobacterium sp. nor by those of Pseudomonas sp., but it was degraded by their mixture. Glyoxylate, a metabolite liberated by the ether-cleaving enzyme, inhibited the growth of the mixed culture. The ether-cleaving enzyme was remarkably inhibited by glyoxylate. Glyoxylate was metabolized faster by Pseudomonas sp. than by Flavobacterium sp., and seemed to be a key material for the symbiosis.     

Secreted β-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 3.2.1.23) at temperatures below 25°C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term stability at temperatures above 25°C.     

Characterization of rhizospheric bacteria isolated from Deschampsia antarctica Desv.

Deschampsia antarctica Desv. is the only gramineae capable of colonizing the Antarctic due to the region’s extreme climate and soil environment. In the present research, bacteria colonizing the rhizospheric soil of D. antarctica were isolated and characterized. The soil studies showed that D. antarctica possesses a wide spectrum of psychrotolerant bacteria with extensive and varied antibiotic resistance, as well as heavy metal tolerance. The bacterial strains isolated from the rhizosphere of D. antarctica also produced a diverse pattern of enzymes. Based on the strain identification with partial characterization of the 16S rRNA gene, the majority of the isolates correspond to different Pseudomonas species, and species of the genus Flavobacterium sp. and Arthrobacter sp. The isolated strains collected from this research constitute a unique collection for future, more detailed taxonomic analysis and physiological characterization, contributing to the search for potential biotechnological uses. These findings and others have great potential for developing new biotechnological products from Antarctic microorganisms.