Biology of budding bacteria

Summary Budding bacteria from aquatic or terrestrial habitats were found to accumulate ferric oxide hydrate (ferric hydroxide) on their cell surfaces. Metal paper clips served as the source of oxidizable iron. Pure cultures deposited ferric hydroxide during growth on sea water medium at a pH of 7.8, but not in a mineral salts medium of normal ionic strength, of pH 7.2, and without NaCl, although some active strains came from fresh water or soil.Ferric iron deposition was found to be initiated at primary active sites on the cell surface; the hyphae and rods eventually become completely encased by the heavy coat.The presence of iron depositing, budding bacteria in fresh water, brackish water or sea water indicates an ubiquitous distribution of these microorganisms.Actively depositing isolates from marine environments are more closely related to Pedomicrobium than to Hyphomicrobium spp. because of their multiple formation of hyphae from rod-shaped swarmer cells. A taxonomic and cultural study of these new forms is in progress.     

Fatty acid composition of various hyphal budding bacteria

The total fatty acid composition of various strains of Hyphomicrobium, Pedomicrobium, and Rhodomicrobium spp. was determined by gas chromatography. In addition, the fatty acid pattern of a new hyphal budding bacterium, strain F-1, was compared with the other patterns obtained. Octadecenoic acid was the main component in most strains, comprising up to 75% of the total fatty acids. Lactobacillic acid and 3-methoxy-tetradecanoic acid were present in varying amounts in the lipids of all organisms except for the new isolate, F-1. This latter strain contained, however, large amounts of iso-heptadecanoic and iso-heptadecenoic acids, not present in the other budding bacteria studied. This composition was consistently found under various culture conditions. The data indicate that, except for the new bacterium F-1, the hyphal budding bacteria studied here are closely related. The total fatty acid composition is thought to be a useful taxonomic criterion for differentiation of these bacteria.     

Isolation and Characterization of a Hyphomicrobium Species and its Polysaccharide Formation from Methanol

A microorganism which could utilize methanol as the sole source of carbon and excreted a new polysaccharide was isolated from soil. This isolate was a stalked bacterium which reproduced by a budding process, and could grow on only methanol, formaldehyde or methylamines as the carbon source. The most suitable nitrogen source for growth was the ammonium ion. The optimum pH and temperature for growth were about 7.0 and 30°C, respectively. The cell growth was inhibited by blue light irradiation. Amino acid composition and fatty acid composition of the cells and electrophoretic behavior of methanol dehydrogenase were also studied. On the basis of these properties as well as taxonomical studies, the isolate (strain JTS-811) was identified as belonging to the genus Hyphomicrobium. This strain had different characteristics as compared to those described for other Hyphomicrobium isolates. At present, it is difficult to give a specific name to this strain, because classification of hyphomicrobia is not clear.     

Form and function in manganese-oxidizing bacteria

Summary Stalked, budding bacteria of the genus Hyphomicrobium are shown to be responsible for manganese deposition in freshwater pipelines. The attachment of the cells to the pipe surface is considered from the point of view of electrostatic attraction and the production of holdfast material. Electrophoretic studies indicate that Hyphomicrobium cells are negatively charged and that their surface ionogenic groups are carboxylic. It is postulated that the curious morphology and mode of reproduction of hyphomicrobia accounts for their efficiency in producing, or coexisting with, the manganese oxides they produce.

---

Zusammenfassung Die gestielte Bakteriengattung der Hyphomikroben wird für die Manganschlammablagerungen in Süßwasserdruckrohrleitungen verantwortlich gemacht. Die Bindung der Zellen an die Rohroberfläche soll durch elektrostatische Anziehungskraft und Bildung von Haftmaterial bewirkt werden. Die elektrophoretischen Versuche zeigen, daß die Hyphomicrobium-Zellen negativ geladen und die Ionen-Gruppen der Oberfläche Carboxyle sind. Es wird postuliert, daß die eigentümliche Morphologie und Vermehrungsweise der Hyphomikroben verantwortlich sind für ihre Fähigkeit, Manganoxyde zu bilden und mit ihnen gemeinsam zu existieren.

     

Cryptic growth within a binary microbial culture

Summary The ability of viable cells of the species Pseudomonas putida and Hyphomicrobium sp. to metabolize the particulate and soluble cellular organic constituents of both species was studied in a series of batch experiments. Both P. putida and Hyphomicrobium sp. were grown in individual batch reactors on either the ¹⁴C-labelled soluble or the particulate debris of sonicated cells of each species derived from steady-state chemostat cultures. Cell generation times (t _g)observed for P. putida cultivated on soluble organic material originating from either sonicated P. putida or Hyphomicrobium sp. cells, were t _g= 2.0 h and t _g= 6.3 h, respectively. Corresponding t _gvalues of Hyphomicrobium sp. on soluble organic material originating from sonicated P. putida and Hyphomicrobium so. were, respectively, 11.6 h and 4.3 h. While particulate debris originating from either species was solubilized by both P. putida and Hyphomicrobium sp., no increases in cell numbers were observed for either species. The data indicate that bacteria are capable of scavenging soluble material released upon cell lysis at near maximal rates; solubilization of debris also occurred but at much slower overall rates with no observable cell replication. The results reaffirm that cryptic growth and turnover of cellular biomass can be significant under situations of low substrate flux or starvation conditions. Offprint requests to: J. D. Bryers     

Identification of a periplasmic dimethylsulphoxide reductase in Hyphomicrobium EG grown under chemolithoheterotrophic conditions with dimethylsulphoxide as carbon source

Hyphomicrobium EG can grow with dimethylsulphoxide as sole carbon and energy source with oxygen as electron acceptor. In the present work we have found that the dimethylsulphoxide reductase of this bacterium could be assayed with dithionite-reduced methylviologen as reductant but not with NADH. Sub-cellular fractionation of Hyphomicrobium EG showed that the dimethylsulphoxide reductase was a periplasmic enzyme. An antibody to the dimethylsulphoxide reductase of Rhodobacter capsulatus cross-reacted with a polypeptide in the periplasmic fraction from Hyphomicrobium EG which had the same M _r as the dimethylsulphoxide reductase of Rhodobacter capsulatus. It is suggested that the reduction of dimethylsulphoxide in Hyphomicrobium involves respiratory electron transfer.Abbreviations DMSO dimethylsulphoxide - DMS dimethylsulphide     

A rapid and specific enrichment procedure forHyphomicrobium spp.

An enrichment procedure for the isolation of stalked bacteria of the genusHyphomicrobium is described. The method is based on the use of an organic C₁ compound as carbon and energy source for growth together with anaerobic incubation in the presence of nitrate as an electron acceptor. Optimal conditions for the growth of a number ofHyphomicrobium isolates have been investigated. Applying these conditions,Hyphomicrobium spp. have been enriched from a wide range of natural habitats within 1–2 weeks. This work was supported by a Grant from the Medical Research Fund, University of Sheffield, England, to M.M.A.     

Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869^T in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Origin of sclerotia-like cells in submerged Claviceps purpurea producing clavine alkaloids

Ultrathin sectioning of submerged mycelium of Claviceps purpurea Tul. producing clavine alkaloids revealed yeast-like budding resulting in asexual sporesblastospores. These deciduous spores were born by extended hyphal cells and retained the same ultrastructure of cell organelles. Both the extended hyphae and the blastospores resembled the cells of ergot sclerotial tissue. A surface culture of C. purpurea Tul. producing no alkaloids was used as a reference.     

Hyphozyma,a new genus of yeast-like Hyphomycetes

Hyphozyma gen. nov. is characterized by pink, slimy colonies, showing initial-or late-stage budding and hyphae which produce conidia in basipetal succession through inconspicuous lateral openings. The cell walls contain rhamnose. Two species and a variety are described and their relation to some genera of yeasts and Hyphomycetes is discussed.     

Peptidase activities during morphogenesis of Mucor racemosus

Multiple peptidase activities are expressed in the dimorphic fungus Mucor racemosus. Peptide hydrolysis was measured using an enzyme-coupled colorimetric assay. Aminopeptidase as well as carboxypeptidase activities increased during spore, swollen spore, and budding yeast-to-hyphae conversions, and activities achieved a maximum level prior to the period of rapid germ tube formation. These increases in peptidase activity were prevented by cycloheximide. Three distinct aminopeptidases (AP) and three distinct carboxypeptidases (CP) were partially purified by gel filtration column chromatography. AP1 (235 kDa), AP2 (112 kDa), and AP3 (70 kDa) were all expressed in spore, yeast, and hyphae. The activity levels of AP2 and AP3 decreased in hyphae entering stationary growth. CP1 (250 kDa) and CP3 (50 kDa) activities were expressed exclusively in hyphae, whereas CP2 (77 kDa) was expressed in spore, yeast, and hyphal forms. CP1 activity was most pronounced in hyphae entering stationary growth. We concluded that M. racemosus expresses a multiplicity of peptidases and that CP1 and CP3 are morphology-specific carboxypeptidases.     

Live-cell imaging elaborating epidermal invasion and vascular proliferation/colonization strategy of Verticillium dahliae in host plants

The soilborne ascomycete fungus Verticillium dahliae causes destructive vascular wilt disease in hundreds of dicotyledonous plant species. However, our understanding of the early invasion from the epidermis to the vasculature and the prompt proliferation and colonization in the xylem tissues remains poor. To elaborate the detailed infection strategy of V. dahliae in host plants, we traced the whole infection process of V. dahliae by live-cell imaging combined with high-resolution scanning electron microscopy. The 4D image series demonstrated that the apex of invading hyphae becomes tapered and directly invades the intercellular space of root epidermal cells at the initial infection. Following successful epidermal invasion, the invading hyphae extend in the intercellular space of the root cortex toward the vascular tissues. Importantly, the high-resolution microscopic and live-cell images demonstrated (a) that conidia are formed via budding at the apex of the hyphae in the xylem vessels to promote systemic propagation vertically, and (b) that the hyphae freely cross adjacent xylem vessels through the intertracheary pits to achieve horizontal colonization. Our findings provide a solid cellular basis for future studies on both intracellular invasion and vascular colonization/proliferation during V. dahliae infection and pathogenesis in host plants.     

Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria

The goal of this study was to identify bacterial populations that assimilated methanol in a denitrifying sequencing batch reactor (SBR), using stable isotope probing (SIP) of ¹³C labeled DNA and quantitatively track changes in these populations upon changing the electron donor from methanol to ethanol in the SBR feed. Based on SIP derived ¹³C 16S rRNA gene clone libraries, dominant SBR methylotrophic bacteria were related to Methyloversatilis spp. and Hyphomicrobium spp. These methylotrophic populations were quantified via newly developed real‐time PCR assays. Upon switching the electron donor from methanol to ethanol, Hyphomicrobium spp. concentrations decreased significantly in accordance with their obligately methylotrophic nutritional mode. In contrast, Methyloversatilis spp. concentrations were relatively unchanged, in accordance with their ability to assimilate both methanol and ethanol. Direct assimilation of ethanol by Methyloversatilis spp. but not Hyphomicrobium spp. was also confirmed via SIP. The reduction in methylotrophic bacterial concentration upon switching to ethanol was paralleled by a significant decrease in the methanol supported denitrification biokinetics of the SBR on nitrate. In sum, the results of this study demonstrate that the metabolic capabilities (methanol assimilation and metabolism) and substrate specificity (obligately or facultatively methylotrophic) of two distinct methylotrophic bacterial populations contributed to their survival or washout in denitrifying bioreactors. Biotechnol. Bioeng. 2009;102: 1527–1536. © 2008 Wiley Periodicals, Inc.     

Experimental infection of white mouse with chrysosporium and paecilomyces

SPF white mice were inoculated intraperitoneally with 5 strains of saprophytic fungi of the mycelial genera Chrysosporium (C. keratinophilum, C. tropicutn) and Paecilomyces (P. lilacinus, P. marquandii, P. victoriae). The fungi caused granulomatous lesions in the peritoneal cavity and they were recultured (except P. lilacinus and P. marquandii) two months after inoculation. Spores, short hyphae and budding cells of all the fungi were observed in the granulomas stained by periodic-acid Schiff (PAS) and methenamine-silver nitrate (Grocott) techniques.     

Manganese(II) adsorption and oxidation by whole cells and a membrane fraction of Pedomicrobium sp. ACM 3067

Heat treatment of Pedomicrobium sp. ACM 3067 enhanced the adsorption of Mn(II) to whole cells but abolished Mn(II)-oxidising activity. In whole cells, optimal Mn(II)-oxidising activity occurred at pH 7 and 25 °C. The apparent K _m of the Mn(II)-oxidising system for Mn(II) was 26 μM. These data confirm that Mn(II) oxidation is an enzymic process in Pedomicrobium sp. ACM 3067. Measurement of Mn(II) oxidation during the growth cycle demonstrated that the highest activity occurred during early- to mid-exponential phase and was independent of the presence of Mn in the growth medium. Mn(II)-oxidising activity was localised to the membrane fraction. Transmission electron microscopy showed that this fraction consisted of double-layered membrane vesicles. Positively charged molecules such as poly-l-lysine interfered with the adsorption and oxidation of Mn(II) by whole cells and membranes. Similarly, aminoglycoside antibiotics such as gentamicin sulfate proved to be potent inhibitors of Mn(II) oxidation. Treatment of cells with the copper chelator diethyldithiocarbamate inhibited Mn(II) oxidation. Enzyme activity was restored by the addition of Cu(II) ions, but not by Co(II) nor Zn(II). We conclude that Mn(II) oxidation in Pedomicrobium sp. ACM 3067 is catalysed by a Cu-dependent enzyme. Received: 14 September 1998 / Accepted: 4 January 1999     

Isolation of a dimethylsulfide-utilizingHyphomicrobium species and its application in biofiltration of polluted air

The methylotrophic bacteriumHyphomicrobium VS was enriched and isolated, using activated sewage sludge as inoculum in mineral medium containing dimethylsulfide (DMS) at a low concentration to prevent toxicity. DMS concentrations above 1 mM proved to be growth inhibiting.Hyphomicrobium VS could use DMS, dimethylsulfoxide (DMSO), methanol, formaldehyde, formate, and methylated amines as carbon and energy source. Carbon was assimilated via the serine pathway. DMS-grown cells respired sulfide, thiosulfate, methanethiol, dimethyldisulfide and dimethyltrisulfide.To testHyphomicrobium VS for application in biofiltration of air polluted with volatile sulfur compounds two laboratory scale trickling biofilters with polyurethane and lava stone as carrier material were started up by inoculation with this bacterium. Both methanol- and DMS-grown cells could be used. Only a short adaptation period was needed. Short term experiments showed that high concentrations of DMS (1–2 µmol 1^–1) were removed very efficiently by the biofilters at space velocities up to 100 h^–1.Abbreviations VSC volatile sulfur compounds - DMS dimethylsulfide - DMDS dimethyldisulfide - DMTS dimethyltrisulfide - MT methanethiol - DMSO dimethylsulfoxide     

Chemostat enrichment and isolation of Hyphomicrobium EG

A stable mixed bacterial culture was obtained by chemostat enrichment using dimethyl-sulphoxide as a carbon and energy source. This culture could not only rapidly oxidize dimethyl-sulphoxide but also dimethyl-sulphide. Enzyme determinations indicated that an important part of it consisted of methylotrophs, which assimilated carbon via the serine pathway. Indeed plate counts revealed the majority of the community to be a Hyphomicrobium species. This organism, designated Hyphomicrobium EG, is an obligate methylotroph which can only grow aerobically on several different C₁-compounds. Its performance on dimethyl-sulphoxide was compared with that of the community and of another recently isolated strain, Hyphomicrobium S. The mixed culture, Hyphomicrobium EG and Hyphomicrobium S had a _max of 0.08, 0.08 and 0.014 h^-1 respectively. The K_S for dimethyl-sulphoxide was the same for all three cultures (3–6 M), whereas that for dimethyl-sulphide of Hyphomicrobium EG after growth on dimethyl-sulphoxide was 3-fold higher than that of the other two cultures (48 and 16 M respectively). After growth on dimethyl-sulphide it improved to 3 M. Dimethyl-sulphide respiration was maximal at a concentration of 100 M; higher concentrations were inhibitory. One of the accompanying organisms, a pink methylotroph, was able to derive energy from the oxidation of thiosulphate. Available cultures of Thiobacillus MS1 that were reported to be able to utilize dimethyl-sulphide could no longer metabolize this compound.     

Genetic manipulation of the restricted facultative methylotroph Hyphomicrobium X by the R-plasmid-mediated introduction of the Escherichia coli pdh genes

The inability of Hyphomicrobium X to grow on compounds such as pyruvate and succinate is most likely due to the absence of a functional pyruvate dehydrogenase (PDH) complex. Further support for this was sought by studying the effect of the introduction of the Escherichia coli pdh genes in Hyphomicrobium X on the pattern of substrate utilization by the latter organism. These genes were cloned by in vivo techniques using the broad-host range conjugative plasmid RP4: :Mucts. Plasmid RP4 derivatives containing pdh genes were selected by their ability to complement a pyruvate dehydrogenase deletion mutant of E. coli, strain JRG746 recA (ace-1pd) 18. The plasmids thus obtained could be transferred through an intermediary host (C600 recA), selecting only for an antibiotic resistance coded for by RP4 and back into JRG746 or other E. coli pdh mutants, upon which they still conferred the wild type phenotype. Enzyme assays showed that the latter strains, when carrying plasmid RP4 pdh1 also possessed PDH complex activity. Conjugation between the auxotrophic E. coli JRG746 (RP4 pdh1) strain and Hyphomicrobium X on pyruvate minimal agar gave rise to progeny which, on the basis of its morphology (stalked bacteria), their ability to grow on C₁-compounds and to denitrify (now also with pyruvate) were identified as hyphomicrobia. This Hyphomicrobium X transconjugant was also able to grow in minimal medium with succinate, but no other novel growth substrates have been identified so far. An analysis of protein extracts with 2-dimensional gel electrophoresis indicated that Hyphomicrobium X and JRG746 only synthesized all three components of the PDH complex when carrying plasmid RP4 pdh1. These results are compatible with the suggested significance of the lack of a functional PDH complex in wild type Hyphomicrobium X.Abbreviations PDH pyruvate dehydrogenase - TCA tricarboxylic acid Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday     

An ultrastructural study of iron and manganese deposition associated with extracellular polymers of pedomicrobium-like budding bacteria

Morphological characteristics of two Pedomicrobium-like budding bacteria are described. A structured surface layer was regularly observed on strain 868. Ruthenium red- and Alcian blue-staining polymers were found on both strains.When either strain was grown in the presence of iron or manganese, the corresponding oxides accumulated on their surfaces. In thin sections iron oxides appeared as fine threads, arrays of particles or dense coatings, depending on the source of iron. Manganese oxides appeared as branching filaments or convoluted ribbons. Both metal oxides stained with ruthenium red. Extraction of the oxides followed by ruthenium red staining revealed that polyanionic polymers previously deposited on the cells were associated with the metals.Treatment of cultures with glutaraldehyde, HgCl₂, or heat, inhibited manganese but not iron deposition, suggesting that iron oxides accumulated by passive, non-biological processes. Manganese oxides apparently accumulated under control of a biological manganese-oxidizing factor. Incomplete inhibition of manganese deposition observed in cell suspensions suggested that, if the oxidizing factor was an enzyme, it was unusually stable.Based on these results, possible mechanisms of iron and manganese deposition in association with extracellular polymers are suggested.