Microcultural Study of Bacterial Size Changes and Microcolony and Ultramicrocolony Formation by Heterotrophic Bacteria in Seawater

With a microculture technique and time-lapse, phase-contrast photomicrography, it was possible to follow the division of individual cells and the development of microcolonies of bacteria in freshly collected marine water samples. A certain number of marine bacteria, upon inoculation onto a nutrient rich agar surface, displayed an increase in size as well as a high growth rate. Other bacteria were identified as very small marine bacteria (ultramicrobacteria). These had a very slow growth rate when inoculated onto a nutrient-rich agar surface. These latter cells formed very small microcolonies (ultramicrocolonies), and cell size did not increase significantly. These two types of marine heterotrophs could be described in terms of zymogenous and autochthonous bacteria, a concept used by Winogradsky for describing soil microorganisms.     

Microscopic examination of natural sessile bacterial populations from an alpine stream.

Natural populations of bacteria assoiciated with the slime on submerged surfaces in a mountain stream were examined by phase-contrast and electron microscopy. The slime contained large numbers of bacteria which were predominantly gram-negative as determined by their cell wall structure. Examination of the in situ distribution of cells revealed that they were enmeshed in an extensive fibrous matrix whose component fibrils were stained with ruthenium red. The arrangement of slime fibrils immediately around individual bacterial cells suggested that this material was produced by these bacteria. This slime facilitated microcolony development and also anchored the bacteria to a particular surface. It is proposed that these slime-enmeshed microcolonies constitute functional communities within which most sessile bacteria live.     

The Bacteroides glycocalyx as visualized by differential interference contrast microscopy

The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.     

马衔山中国沙棘根瘤内共生细菌多样性研究

采集甘肃省兰州市马衔山中国沙棘(Hippophae rhamnoidoes)根瘤样品,采用高通量测序和纯培养方法,分析中国沙棘根瘤内定殖的内共生细菌的组成、相对丰度和物种多样性,并比较两种方法研究结果的差异。在不同的微生物分类单元,高通量测序检测到中国沙棘根瘤内共生细菌24门50纲90目167科215属;而纯培养方法仅检测到3门5纲7目8科8属。进一步分析沙棘根瘤内共生细菌主要类群的相对丰度,发现高通量测序与纯培养方法的结果有明显差异,在科和属的分类单元上差异尤其显著。两种方法都表明中国沙棘根瘤内共生细菌具有丰富的多样性,但高通量测序能够较为全面的反映中国沙棘根瘤内共生细菌的群落组成,而纯培养方法仅能够分离到部分可培养的中国沙棘根瘤内共生细菌,在很大程度上极可能低估中国沙棘根瘤内共生细菌的物种组成并高估其丰度。     

Aerobic Heterotrophic Bacteria Indigenous to pH 2.8 Acid Mine Water: Microscopic Examination of Acid Streamers

"Acid streamers" found in acid coal mine drainage consist of bacteria trapped within an extracellular fibrillar polymer network. Inorganic compounds also precipitate within the polymer network. Several bacteria which appear to be different and are presumed to be different species are associated in the slimy mass of the "acid streamers." The "streamers" contain individual microcolonies or microcosms that can be recognized by a selective polysaccharide stain, which suggests that the slime streamer is a conglomeration of polymers produced by more than one species.     

Fine structure of nitrogen-fixing leguminous root nodules from the Canadian Arctic

Effective (nitrogen-fixing) root nodules of Oxytropis maydelliana Trautv., O. arctobia Bunge and Astragulus alpinus L. were collected in the high Arctic tundra and subsequently processed for structural studies. The cylindrically-shaped perennial nodules consisted of the following tissues: nodule cortex, nodule meristem, nodular vascular bundles, an active central region with uninfected and infected cells at various stages of development, and a proximal region of senescent cells. The active central region was dark red-coloured due to the presence of the pigment leghemoglobin. The host cells became infected by the growth of infection threads into cells recently derived from the nodule meristem and the subsequent endocytotic release of rhizobia from unwalled membrane-bound regions of the infection thread. The host plasma membrane adjacent to the unwalled regions of infection thread gave rise to the peribacteroid membrane which surrounded the released bacteria. Thus, nodule development and the basic tissue arrangement of the arctic nodules was similar to that of cylindrically-shaped nodules formed on temperate species of legumes.
The arctic legume nodules are unique in having large numbers of lipid droplets present in the cytoplasm of the nodule cortex and uninfected cells of the central active region. Newly infected cells also have lipid droplets. More developed infected cells lack lipid droplets but often contain amyloplasts. Mature differentiated bacteria were spherically-shaped and contained electron-dense inclusions. Electron-dense material was also present in vesicles formed from dilated endoplasmic reticulum and in the peribacteroid space. The lipid droplets present in the host cytoplasm of the nodule cortex and uninfected cells of the central tissue may be storage products which are used to support nitrogen-fixation in nodules growing under cool temperatures of this harsh environment.     

浙江温州水稻根瘤状组织的结构分析

用光镜、扫描电镜和透射电镜观察的方法,对浙江温州水稻根上的瘤状组织进行了结构分析,并与豆科植物根瘤的结构进行了比较。壮苗期水稻的瘤状组织内皮层少数薄壁细胞内有细菌存在,但细胞有防御反应,细胞质凝聚和纤维化,分不清细胞质的精细结构,说明细胞已经死亡;孕穗抽穗期水稻根瘤状组织,表皮黑褐色,木栓化。外皮层内无维管组织,内皮层薄壁细胞膨大,但没有含菌细胞。有的瘤状组织内外皮层细胞基本上木质化。在内皮层薄壁细胞内有齿轮状的特殊结构。光镜和扫描电镜观察到的丝状物和珊瑚状花纹的球体,实为某种真菌的菌丝和孢子。因此,浙江温州的水稻根瘤状组织的结构与豆科根瘤完全不同,实为混有细菌和真菌的愈伤组织     

Bacteriology of Manganese Nodules: I. Bacterial Action on Manganese in Nodule Enrichments

Bacteria, found in manganese nodules from the Atlantic Ocean, enhance the adsorption of Mn from sea water by crushed manganese nodules in the presence of peptone. When bacterial outgrowth from crushed manganese nodules was experimentally delayed, peptone did not enhance Mn adsorption by nodular substance, but hindered it in some cases. A mechanism to explain the role of bacteria in enhancing Mn adsorption by manganese nodules is presented. Oyster shells were shown to adsorb Mn in the absence of bacteria. Peptone did not enhance the rate of Mn adsorption. Adsorbed Mn was not visibly oxidized during experimental observation. These results suggest one way whereby nodule formation may be initiated in the oceans. Some bacteria in the nodules were found to release manganese from them in the presence of glucose and peptone. Bacteria may, therefore, play a role not only in nodule buildup but also in nodule breakdown.     

Role of Motility and Chemotaxis in Efficiency of Nodulation by Rhizobium meliloti

Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants was able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation in the initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10⁵ to 10⁶ bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants. The mutants were also somewhat less effective than their parent as competitors in root adsorption assays. It appears that motility and chemotaxis are quantitatively important traits that facilitate the initial contact and adsorption of symbiotic rhizobia to the host root surface, increase the efficiency of nodule initiation, and increase the rate of infection development.     

Lipopolysaccharide epitope expression of Rhizobium bacteroids as revealed by in situ immunolabelling of pea root nodule sections.

To investigate the in situ expression of lipopolysaccharide (LPS) epitopes on nodule bacteria of Rhizobium leguminosarum, monoclonal antibodies recognizing LPS macromolecules were used for immunocytochemical staining of pea nodule tissue. Many LPS epitopes were constitutively expressed, and the corresponding antibodies reacted in nodule sections with bacteria at all stages of tissue infection and cell invasion. Some antibodies, however, recognized epitopes that were only expressed in particular regions of the nodule. Two general patterns of regulated LPS epitope expression could be distinguished on longitudinal sections of nodules. A radial pattern probably reflected the local physiological conditions experienced by endosymbiotic bacteria as a result of oxygen diffusion into the nodule tissue. The other pattern of expression, which followed a linear axis of symmetry along a longitudinal section of the pea nodule, was apparently associated with the differentiation of nodule bacteria and the development of the nitrogen-fixing capacity in bacteroids. Basically similar patterns of LPS epitope expression were observed for pea nodules harboring either of two immunologically distinct strains of R. leguminosarum bv. viciae, although these epitopes were recognized by different sets of strain-specific monoclonal antibodies. Furthermore, LPS epitope expression of rhizobia in pea nodules was compared with that of equivalent strains in nodules of French bean (Phaseolus vulgaris). From these observations, it is suggested that structural modifications of Rhizobium LPS may play an important role in the adaptation of endosymbiotic rhizobia to the surrounding microenvironment.     

Ultrastructure of Holothuria polii encapsulating body

The coelomic cavity of freshly collected Holothuria polii specimens contains a variable number of brown pigmented and unpigmented encapsulating structures. They are composed of nodules with entrapped parasites and an internodular mass which comprises a number of nodules. Both brown and unpigmented bodies occur in different size classes depending upon the number of nodules accumulated in a complete body. The unpigmented bodies probably represent an early unmelanized stage of the brown ones.
The nodule was ultrastructurally constituted by foreign bodies surrounded by a fibrous, electron-dense, non-cellular layer, probably melanin, followed by a layer of elongated and extremely flattened amoebocytes. Nodules were assembled in an internodular mass formed by amoebocytes type I, II and III spherule cells. As for the amoebocytes constituting the nodule, those present in the spaces between nodules did not develop junctional complexes. It is conceivable that the intricate network established among the cell processes could represent the mechanical force maintaining the whole structure.
As suggested by our results, two functional amoebocyte populations seem to be responsible for the organization of the scavenger body: 1. encapsulating amoebocytes, characteristically non phagocytosing, elongated cells; and 2, phagocytosing amoebocytes. The former organize the nodules, the latter constitute the internodular mass of several nodules.
Most probably, the double scavenger activity justifies the considerable complexity of the H. polii encapsulating structure compared to other invertebrates.
Spherule cells participate only in constituting the internodular mass.     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.     

In situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry

Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established. In this procedure, plant nodules were considered as natural bioreactors that amplify clonal populations of nitrogen-fixing bacteroids. Following a simple isolation procedure, bacteroids were fingerprinted by analysing biomarker cellular proteins of 3 to 13 kDa using Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry. In total, bacteroids of more than 1,200 nodules collected from roots of three legumes of the Phaseoleae tribe (cowpea, soybean or siratro) were examined. Plants were inoculated with pure cultures of a slow-growing Bradyrhizobium japonicum strain G49, or either of two closely related and fast-growing Sinorhizobium fredii strains NGR234 and USDA257, or with mixed inoculants. In the fully automatic mode, correct identification of bacteroids was obtained for >97% of the nodules, and reached 100% with a minimal manual input in processing of spectra. These results showed that MALDI-TOF MS is a powerful tool for the identification of intracellular bacteria taken directly from plant tissues.     

Studies on the Physiology of Nodule Formation: III. Experiments on the Excision of Root-tips and Nodules

The excision of nodules and root-tips from red clover inoculatedwith an effective strain of nodule bacteria leads to an increasein the number of nodules subsequently formed. This is thoughtto be due to the removal by excision of the source of an inhibitor.In the nodule the inhibitory activity appears to be centredin the growing-point and not in the bacterial tissue, sincethe excision of the nodular meristem only will stimulate furthernodulation. The response to excision depends upon the numberof excisions made and is inversely related to the inherent susceptibilityof the individual plant. Excision of nodules from plants inoculated with ineffectivestrains of bacteria has no influence on further nodulation,presumably because the growing-points of the nodules are abortive.     

Hydrocarbon utilization by nodule bacteria and plant growth-promoting rhizobacteria

Standard and locally isolated nodule bacteria and plant growth-promoting rhizobacteria (PGPR) were grown on crude oil and individual pure hydrocarbons as sole sources of carbon and energy. The nodule bacteria included two standard Rhizobium leguminosarum strains, two standard Bradyrhizobium japonicum strains, and one unknown nodule bacterial strain that was locally isolated from Vicia faba nodules. The PGPR included one standard Serratia liquefaciens strain and two locally isolated strains of Pseudomonas aeruginosa and Flavobacterium sp. The pure hydrocarbons tested included n-alkanes with chain lengths from C9 to C40 and the aromatic hydrocarbons benzene, biphenyle, naphthalene, phenanthrene, and toluene. Quantitative gas liquid chromatographic analyses confirmed that pure cultures of representative nodule bacteria and PGPR could attenuate n-octadecane and phenanthrene in the surrounding nutrient medium. Further, intact nodules of V. faba containing bacteria immobilized on and within those nodules reduced hydrocarbon levels in a medium in which those nodules were shaken. It was concluded that legume crops are suitable phytoremediation tools for oily soil, since they enrich such soils not only with fixed nitrogen, but also with hydrocarbon-utilizing microorganisms. Further, legume nodules may have biotechnological value as materials for cleaning oily liquid wastes.     

Type IV Pilin Post-Translational Modifications Modulate Material Properties of Bacterial Colonies

Bacterial type 4 pili (T4P) are extracellular polymers that initiate the formation of microcolonies and biofilms. T4P continuously elongate and retract. These pilus dynamics crucially affect the local order, shape, and fluidity of microcolonies. The major pilin subunit of the T4P bears multiple post-translational modifications. By interfering with different steps of the pilin glycosylation and phosphoform modification pathways, we investigated the effect of pilin post-translational modification on the shape and dynamics of microcolonies formed by Neisseria gonorrhoeae. Deleting the phosphotransferase responsible for phosphoethanolamine modification at residue serine 68 inhibits shape relaxations of microcolonies after perturbation and causes bacteria carrying the phosphoform modification to segregate to the surface of mixed colonies. We relate these mesoscopic phenotypes to increased attractive forces generated by T4P between cells. Moreover, by deleting genes responsible for the pilin glycan structure, we show that the number of saccharides attached at residue serine 63 affects the ratio between surface tension and viscosity and cause sorting between bacteria carrying different pilin glycoforms. We conclude that different pilin post-translational modifications moderately affect the attractive forces between bacteria but have severe effects on the material properties of microcolonies.     

Ontogeny and ultrastructure of spontaneous nodules in alfalfa (Medicago sativa)

Summary The development of spontaneous nodules, formed in the absence ofRhizobium and combined nitrogen, on alfalfa (Medicago sativa L. cv. Vernal) was investigated at the light and electron microscopic level and compared to that ofRhizobium-induced normal nodules. Spontaneous nodules were initiated from cortical cell divisions in the inner cortex next to the endodermis, i.e., the site of normal nodule development. These nodules, on uninoculated roots, were white multilobed structures, histologically composed of nodule meristems, cortex, endodermis, central zone and vascular strands. Nodules were devoid of intercellular or intracellular bacteria confirming microbiological tests. Early development of spontaneous nodules was initiated by series of anticlinal followed by periclinal divisions of dedifferentiated cells in the inner cortex of the root. These cells formed the nodular meristem from which the nodule developed. The cells in the nodule meristems divided unequally and differentiated into two distinct cell types, one larger type being filled with numerous membrane-bound starch grains, and the other smaller type with very few starch grains. There were no infection threads or bacteria in the spontaneous nodules at any stage of development. This size differentiation is suggestive of the different cell sizes seen inRhizobium-induced nodules, where the larger cell type harbours the invading bacteria and the smaller type is essential in supportive metabolic roles. The ontogenic studies further support the claim that these structures are nodules rather than aberrant lateral roots, and that plant possess all the genetic information needed to develop a nodule with distinct cell types. Our results suggest that bacteria and therefore theirnod genes are not necessarily involved in the ontogeny and morphogenesis of spontaneous and normal nodules in alfalfa.Abbreviations EH smallest emergent root hair - EM electron microscope - enod2 early nodulin2 gene - RT root tip - RER rough endoplasmic reticulum - YEMG yeast extract-mannitol-gluconate     

Nodule distribution on the roots of actinorhizal Discaria trinervis (Rhamnaceae) growing in pots

Roots of actinorhizal plants can develop nitrogen-fixing nodules with actinomycetic bacteria of the genus Frankia. We aimed to know if unrestricted growth of roots in pots could influence the pattern of nodule development that we had previously observed for Discaria trinervis growing in pouches. Growth pouches, although being a space saving device convenient for the analysis of nodule development, do restrict root growth. Thus, the pattern of root nodule development was analysed in actinorhizal D. trinervis growing in pots with inert substrates. Inoculation of axenic seedlings growing in perlite resulted in clustering of nodules in a defined region of the taproot and upper lateral roots. When surface sterilized seeds were sown in pots containing vermiculite that had been previously inoculated with Frankia cells, nodules were again concentrated in defined portions of the main and lateral roots. Potted plants developed comparable numbers of nodules with respect to plants grown in pouches. However, a significant proportion of nodules appeared in lateral roots. As it was first inferred from field grown plants, these results confirm that D. trinervis plants growing in pots display the same autoregulatory mechanism for nodule formation that was previously observed in growth pouches.