Conti, S. F. (Dartmouth Medical School, Hanover, N.H.), and Peter Hirsch. Biology of budding bacteria. III. Fine structure of Rhodomicrobium and Hyphomicrobium spp. J. Bacteriol. 89:503–512. 1965.—The ultrastructure of 14 strains of hyphomicrobia, and of Rhodomicrobium vannielii, was investigated by means of electron microscopy of thin sections. The majority of the strains of hyphomicrobia possessed a well-developed internal membrane system, which appeared to be derived by invagination from the cytoplasmic membrane. The subcellular organization of the hyphomicrobia and R. vannielii was investigated.
Cells of Rhodomicrobium vannielii were grown in a controlled environment at several different light intensities. Differential rates of bacteriochlorophyll (BChl) synthesis and specific BChl contents were inversely related to the light intensity. On the other hand, the specific rate of growth—before reaching a maximal value—was directly related to the intensity of the light. Thin sections of cells grown at moderately low light showed the typical peripherally located, symmetrically distributed lamellate system, whereas an asymmetrical distribution of a less extensive lamellate system occurred in cells grown at high light intensities. It is proposed that a limited number of individual units of the lamellate system are originally derived from inward folds of the cytoplasmic membrane, and that subsequent lamellae arise by proliferation, including possible forking and definite folding back, of the few original lamellar membranes.
The phototrophic purple non-sulfur bacterium Rhodomicrobium vannielii grew phototrophically (illuminated anaerobic conditions) on a variety of aromatic compounds (in the presence of CO2). Benzoate was universally photocatabolized by all five strains of R. vannielii examined, and benzyl alcohol was photocatabolized by four of the five strains. Catabolism of benzyl alcohol by phototrophic bacteria has not been previously reported. Other aromatic substrates supporting reasonably good growth of R. vannielii strains were the methoxylated benzoate derivatives vanillate (4-hydroxy-3-methoxybenzoate) and syringate (4-hydroxy-3,5-dimethoxybenzoate). However, catabolism of vanillate and syringate led to significant inhibition of bacteriochlorophyll synthesis in R. vannielii cells, eventually causing cultures to cease growing. No such effect on photopigment synthesis in cells grown on benzoate or benzyl alcohol was observed. Along with a handful of other species of anoxygenic phototrophic bacteria, the ability of the species R. vannielii to photocatabolize aromatic compounds indicates that this organism may also be ecologically significant as a consumer of aromatic derivatives in illuminated anaerobic habitats in nature.
Hopanoids are present in vast amounts as integral components of bacteria and plants with their primary function to strengthen rigidity of the plasma membrane. To establish their roles more precisely, we conducted sequencing of the whole genome of Rhodomicrobium udaipurense JA643T isolated from a fresh water stream of Udaipur in Himachal Pradesh, India, by using the Illumina HiSeq pair end chemistry of 2 × 100 bp platform. Determined genome showed a high degree of similarity to the genome of R. vannielii ATCC17100T and the 13.7 million reads generated a sequence of 3,649,277 bp possessing 3,611 putative genes. The genomic data were subsequently investigated with respect to genes involved in various features. The machinery required for the degradation of aromatic compounds and resistance to solvents as well as all that required for photosynthesis are present in this organism. Also, through extensive functional annotation, 18 genes involved in the biosynthesis of hopanoids are predicted, namely those responsible for the synthesis of diploptene, diplopterol, adenosylhopane, ribosylhopane, aminobacteriohopanetriol, glycosyl group containing hopanoids and unsaturated hopanoids. The hopanoid biosynthetic pathway was then inferred based on the genes identified and through experimental validation of individual hopanoid molecules. The genome data of R. udaipurense JA643T will be useful in understanding the functional features of hopanoids in this bacterium.
Rhodomicrobium udaipurense JA643T; genome sequence; Illumina Hiseq; hopanoid biosynthesis pathway
Eight components, seven of which contained phosphorus, were found in the phospholipid fraction of Rhodomicrobium vannielii. The major components were lipoamino acid (o-ornithine ester of phosphatidyl glycerol, 46.5%) and phosphatidyl choline (26.5%). The other six components were phosphatidyl glycerol (9.7%), bisphosphatidic acid (6.7%), phosphatidyl ethanolamine (4.5%), phosphatidic acid (1.8%), lysophosphatidyl glycerol-o-ornithine ester (3.2%), and N,N-ornithine amide of unidentified fatty acid (0.95%). Total phospholipid accounted for 4.2% of cell dry weight. The major fatty acid was vaccenic acid, C18:1, which accounted for approximately 90% of the total fatty acids of the complex lipid fraction. The other four fatty acids were C16:0 (6.25%), C18:0 (3.8%), C14:0 (0.7%), and C16:1 (0.35%). The sulfolipid content was 0.01% of the cell dry weight or 0.14 μmoles per g of dried cells, assuming that its fatty acid component is vaccenic acid. No steroids were detected.
The fine structure of the photosynthetic bacterium Rhodomicrobium vannielii was studied by the ultra thin sectioning technique. Cells were fixed in buffered osmium tetroxide and embedded in Epoxy resin. The feature most common to nearly all cells was an array of intracellular membranes situated in a concentric manner at the periphery of the cell. The membranes were mostly paired and quite often five pairs were seen aligned together. Calculations from densitometric tracings showed the average width of a "unit" membrane to be 65 A. Sections of material from disrupted cells after passage through a sucrose gradient revealed vesicular forms composed of membranes similar in width to those in the intact cell. Absorption spectra of both intact cells and isolated membranes were very similar in the bacteriochlorophyll regions. Septa and membranes were demonstrated in the filaments that join mature cells. No evidence for chromatophores was obtained although the methods used were adequate for their demonstration in Rhodospirillum rubrum.
Under appropriate cultural conditions, cell suspensions of Rhodomicrobium vannielii effect two distinct photoreactions involving molecular hydrogen: (i) the photoreduction of carbon dioxide, and (ii) the photoproduction of hydrogen.
Cells of Rhodomicrobium vannielii grown at 29 C in a lactate-containing medium were extracted at room temperature with organic solvents. The extractable fraction contained the bulk of the simple lipid (1.87% of cell dry weight) and complex lipids (phospholipids, 4.2%; sulfolipid, 0.01%), coenzyme Q (0.09%), and pigments (carotenoids 1.2%; bacteriochlorophyll, 1.9%). The cell residue contained the bound lipids (nonpolar fatty acid fraction, 1.86%; polar hydroxy fatty acids, 0.49%). The residue also contained poly-β-hydroxybutyric acid (0.2%), which was extracted in boiling chloroform. In both the simple and complex lipids, vaccenic acid (11-octadecenoic acid) was the largest single component (approximately 90% in each fraction). The fatty acids of the bound lipid contained 35% vaccenic acid, even- and odd-numbered saturated and unsaturated straight-chain fatty acids, cyclopropane-, branched-, and α- and β-hydroxy fatty acids. The extractable lipids contained only straight-chain saturated and unsaturated even-numbered fatty acids. Nearly 60% of hydroxy fatty acid fraction was α-hydroxydodecanoic acid (24%) and β-hydroxydodecanoic acid (34.5%). Coenzyme Q was crystallized and identified as Q9 on the basis of melting point and chromatographic properties. Q10 had been previously reported.
Water samples were collected aseptically several times throughout the year at nine stations on the Red Cedar River, a stream flowing through farmland and receiving effluent from several municipalities in central Michigan. Total prosthecate bacteria were enumerated by both direct and viable counting techniques. By direct techniques, these bacteria accounted for 0.62 to 1.1% of the total microflora during the study. The predominant type of appendaged bacteria was the caulobacters (Caulobacter, Asticcacaulis, and the fusiform caulobacter), which accounted for 64 to 93% of the total prosthecate forms. The others of importance were prosthecomicrobia (< 1 to 24%), including Prosthecomicrobium and Prosthecochloris; hyphomicrobia (< 1 to 15%), including Hyphomicrobium and Rhodomicrobium; and Ancalomicrobium (< 1 to 6%). The viable counts of heterotrophs also indicated that the caulobacters were the most numerous prosthecate bacteria in the stream. They ranged from fewer than 1 per ml to a maximum of almost 4,000 per ml. During the coldest period, when the total viable counts decreased to about 104 per ml compared to their summer high of over 107 per ml, the caulobacters actually increased in numbers. In December (temperature 0 to 1 C), they comprised from 0.09 to 1.0% of the viable microbial count, and in March (6.0 to 8.0 C) they accounted for 0.14 to 2.8%. The other heterotrophic prosthecate bacteria were generally found at numbers less than 1 per ml, with the exception of the December study when Hyphomicrobium was present in numbers as high as 2,400 per ml. There was no consistent correlation between the frequency of prosthecate bacteria and total coliforms in the stream during the investigation.
The genetic relatedness of a number of budding and prosthecate bacteria was determined by deoxyribonucleic acid (DNA) homology experiments of the direct binding type. Strains of Hyphomicrobium sp. isolated from aquatic habitats were found to have relatedness values ranging from 9 to 70% with strain “EA-617,” a subculture of the Hyphomicrobium isolated by Mevius from river water. Strains obtained from soil enrichments had lower values with EA-617, ranging from 3 to 5%. Very little or no homology was detected between the amino acid-utilizing strain Hyphomicrobium neptunium and other Hyphomicrobium strains, although significant homology was observed with the two Hyphomonas strains examined. No homology could be detected between prosthecate bacteria of the genera Rhodomicrobium, Prosthecomicrobium, Ancalomicrobium, or Caulobacter, and Hyphomicrobium strain EA-617 or H. neptunium LE-670. The grouping of Hyphomicrobium strains by their relatedness values agrees well with a grouping according to the base composition of their DNA species. It is concluded that bacteria possessing cellular extensions represent a widely diverse group of organisms.
We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.
Beggiatoa; budding; epibiont; sulfur bacteria; Thiomargarita; Thioploca
Prosthecate bacteria comprised 0.6 to 10.5% of the bacterial community in samples from 11 pulp mill waste aeration lagoons. Because of their distinct morphology, the genera Ancalomicrobium, Caulobacter, Prosthecobacter, Prosthecomicrobium, Stella, and Hyphomicrobium or Hyphomonas could be identified and enumerated by direct microscopic examination. Monthly samples from one lagoon showed that several genera varied from undetectable to predominant among the appendaged organisms. Temperature (season), type of wood pulped, and pulping process did not significantly affect the density of prosthecate bacteria.
A microscopic survey is presented of the most commonly observed and morphologically conspicuous microorganisms found attached to natural surfaces or to artificial materials deposited in the immediate vicinity of thermal submarine vents at the Galapagos Rift ocean spreading zone at a depth of 2,550 meters. Of special interest were the following findings: (i) all surfaces intermittently exposed to H2S-containing hydrothermal fluid were covered by layers, ca. 5 to 10 μm thick, of procaryotic, gram-negative cells interspaced with amorphous metal (Mn-Fe) deposits; (ii) although some of the cells were encased by dense metal deposits, there was little apparent correlation between metal deposition and the occurrence of microbial mats, (iii) highly differentiated forms appeared to be analogues of certain cyanobacteria, (iv) isolates from massive mats of a prosthecate bacterium could be identified as Hyphomicrobium spp., (v) intracellular membrane systems similar to those found in methylotrophic and nitrifying bacteria were observed in approximately 20% of the cells composing the mats, (vi) thiosulfate enrichments made from mat material resulted in isolations of different types of sulfur-oxidizing bacteria including the obligately chemolithotrophic genus Thiomicrospira.
Caulobacters are prosthecate (stalked) bacteria that elaborate an attachment organelle called a holdfast at the tip of the cellular stalk. We examined the binding of lectins to the holdfasts of 16 marine Caulobacter strains and 10 freshwater species or strains by using a panel of fluorescein-conjugated lectins and fluorescence microscopy. The holdfasts of all the marine isolates bound to only wheat germ agglutinin (WGA) and other lectins that bind N-acetylglucosamine (GlcNac) residues. The freshwater caulobacters showed more variability in holdfast composition. Some bound only to WGA and comparable lectins as the marine strains did. Others bound additional or other lectins, and some did not bind to the lectins tested. The binding of WGA appeared to involve the regions of the holdfast involved with adhesion; a holdfast bound to WGA was significantly less adhesive to glass. Competition experiments with WGA-binding holdfasts and oligomers of GlcNac demonstrated that trimers of GlcNac (the preferred substrate for WGA binding) were more effective than dimers or monomers in preventing WGA binding to holdfasts, suggesting that stretches of contiguous GlcNac residues occur in the WGA-binding holdfasts. In addition, differences between freshwater and marine holdfasts in the strength of WGA binding were noted. The effect of a number of proteolytic and glycolytic enzymes on holdfast integrity was examined; the proteases had no effect for all caulobacters. None of the glycolytic enzymes had an effect on marine caulobacter holdfasts, but chitinase and lysozyme (both attack oligomers of GlcNac) disrupted the holdfasts of those freshwater caulobacters that bound WGA. Despite some similarity to chitin, holdfasts did not bind Calcofluor and no measurable effects on holdfast production were detectable after cell growth in the presence of diflubenzuron or polyoxin D, inhibitors of chitin synthesis in other systems. Finally, the holdfasts of all caulobacters bound to colloidal gold particles, without regard to the coating used to stabilize the gold particles. This binding was stronger or more specific than WGA binding; treatment with colloidal gold particles prevented WGA binding, but the reverse was not the case.
The citric acid cycle enzyme malate dehydrogenase was purified to homogeneity from the nonsulfur purple bacteria Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodomicrobium vannielii, and Rhodocyclus purpureus. Malate dehydrogenase was purified from each species by either a single- or a two-step protocol: triazine dye affinity chromatography was the key step in purification of malate dehydrogenase in all cases. Purification of malate dehydrogenase resulted in a 130- to 240-fold increase in malate dehydrogenase specific activity, depending on the species, with recoveries ranging from 30 to 70%. Homogeneity of malate dehydrogenase preparations from the four organisms was determined by sodium dodecyl sulfate and nondenaturing polyacrylamide gel electrophoresis; a single protein band was observed in purified preparations by both techniques. The molecular weight of native malate dehydrogenases was determined by four independent methods and estimated to be in the range of 130,000 to 140,000 for the enzyme from R. capsulatus, R. rubrum, and R. vannielii and 57,000 for that from R. purpureus. It is concluded that malate dehydrogenase from R. capsulatus, R. rubrum, and R. vannielii is a tetramer composed of four identical subunits, while the enzyme from R. purpureus is a dimer composed of two identical subunits.
Hyphomonas, a genus of budding, prosthecate bacteria, are primarily found in the marine environment. Seven type strains, and 35 strains from our collections of Hyphomonas, isolated from the Pacific Ocean, Atlantic Ocean, Arctic Ocean, South China Sea and the Baltic Sea, were investigated in this study using multilocus sequence analysis (MLSA). The phylogenetic structure of these bacteria was evaluated using the 16S rRNA gene, and five housekeeping genes (leuA, clpA, pyrH, gatA and rpoD) as well as their concatenated sequences. Our results showed that each housekeeping gene and the concatenated gene sequence all yield a higher taxonomic resolution than the 16S rRNA gene. The 42 strains assorted into 12 groups. Each group represents an independent species, which was confirmed by virtual DNA-DNA hybridization (DDH) estimated from draft genome sequences. Hyphomonas MLSA interspecies and intraspecies boundaries ranged from 93.3% to 96.3%, similarity calculated using a combined DDH and MLSA approach. Furthermore, six novel species (groups I, II, III, IV, V and XII) of the genus Hyphomonas exist, based on sequence similarities of the MLSA and DDH values. Additionally, we propose that the leuA gene (93.0% sequence similarity across our dataset) alone could be used as a fast and practical means for identifying species within Hyphomonas. Finally, Hyphomonas' geographic distribution shows that strains from the same area tend to cluster together as discrete species. This study provides a framework for the discrimination and phylogenetic analysis of the genus Hyphomonas for the first time, and will contribute to a more thorough understanding of the biological and ecological roles of this genus.
The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.