Oligotrophic bacterioplankton with a novel single-cell life strategy

A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4',6'-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 micro g of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells.     

Oligotrophic Bacterioplankton with a Novel Single-Cell Life Strategy

A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4′,6′-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 μg of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells.     

Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique

Abstract Kanamycin-resistant Pseudomonas fluorescens DF57-3 cells (Tn5 modified) inoculated in soil microcosms rapidly lost their culturability, as defined by visible colony formation on Kings B agar supplemented with kanamycin. Thus, after 40 days only 0.02–0.35% of the initial inoculum was culturable. A microcolony epifluorescence technique was developed to determine the viable, but non-culturable subpopulation. A suspension of bacteria from the soil was prepared in salt solution after a sonication procedure and a sample was filtered onto a 0.2 μm Nuclepore filter. The filter was then placed for 3–4 days on the surface of Kings B agar before staining with acridine orange for epifluorescence microscopy. By staining and washing the filters carefully, disruption of microcolonies could be avoided. A majority of the microcolonies resulted from 2–3 cell divisions during the first 2 days of the incubation period, after which the cell divisions stopped. These microcolonies were taken to represent a population of viable, but non-culturable cells and comprised about 20% of the initial inoculum. A similar recovery was obtained when the filters were incubated on the surface of citrate minimal medium or soil extract medium. A few microcolonies showed continued growth on the filters, however, and their number corresponded well with that of visible macrocolonies. Observation by microscopy of a few (2–3) cell divisions (microcolony epifluorescence technique) is proposed for determination of subpopulations of viable, but non-culturable bacteria in soil.     

A method for estimating viability of aquatic bacteria by slide culture

To estimate the viability of freshwater bacteria, slide cultures were prepared by spreading 10 μl of water, concentrated by centrifugation, over 1 cm² of agar. After drying, a cover-slip was placed on the agar. Following incubation, microcolonies and single cells were counted under a phase contrast microscope, and the viability estimated. Incubation at 10°C on casein-peptone-starch agar for 24 or 72 h provided the highest estimates of viability ( ca 45%). Most microcolonies developed under these conditions and the total number of microcolonies and single cells did not decrease significantly during incubation. Several morphological types of bacteria were observed as both microcolonies and single cells. Most microcolonies consisted of two cells, although some larger ones were present. The method could be used as an alternative to the spread plate and other methods which assess viability from bacterial growth on agar.     

Viability of Indigenous Soil Bacteria Assayed by Respiratory Activity and Growth

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The effect on viable and culturable cell numbers of a temperature increase from 4 to 17°C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, affected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.     

A critical stage in the formation of acid mine drainage: Colonization of pyrite by Acidithiobacillus ferrooxidans under pH-neutral conditions

A dominant Acidithiobacillus ferrooxidans ssp. was isolated from the supergene copper deposit in Morenci, Arizona, USA. Washed bacterial suspensions (10⁸ MPN per treatment), in pH‐neutral buffer, were inoculated onto pyrite cubes for 24 h. Heterogeneous bacterial absorption onto the pyrite removed approximately 90% of the viable bacteria from the inoculum. At T = 0, the bacteria were observed primarily in regions enriched in phosphorus. Over 30 days, the bacterial population on the pyrite cubes increased from 1.3 × 10⁷ to 2.9 × 10⁸ bacteria cm^?2. During this growth stage, low levels of thiobacilli (228 ± 167 MPN mL^?1) were also recovered from the fluid phase; however, this population decreased to zero within 30 days. Growth on pyrite occurred as micrometre‐scale planar microcolonies, a biofilm, coating the mineral surfaces. These microcolonies possessed viable thiobacilli, even after 4 months at ‘circumneutral pH’. Imaging the pyrite cubes using SEM‐EDS and scanning force microscopy demonstrated that the thiobacilli grew as iron oxy‐hydroxide‐cemented cells, leading to the formation of mineralized microcolonies. Removing the iron oxy‐hydroxides with oxalic acid did not dislodge the bacteria, demonstrating that the secondary minerals were not responsible for ‘gluing’ the bacteria to the pyrite surface. Removing organic material, i.e. the cells, by an oxygen plasma treatment revealed the presence of corrosion pits the size and shape of bacteria. Because of the inherent geochemical constraints on pyrite oxidation at neutral pH, the colonization of pyrite under circumneutral pH conditions must be facilitated by the development of an acidic nanoenvironment between the bacteria and the pyrite mineral surface.     

Escherichia coli K-12 cell-cell interactions seen by time-lapse video.

The high degree of organization in mature bacterial colonies suggests specific interactions between the cells during colony development. We have used time-lapse video microscopy to find evidence for cell-cell interactions. In its initial stages, Escherichia coli K-12 colony morphogenesis displayed control of the geometry of cell growth and involved intimate side-by-side associations. When microcolonies developed from isolated single bacteria, a directed process of elongation and division resulted in the appearance of a symmetrical four-cell array. When growth began with separate but nearby bacteria, the daughters of different cells elongated towards each other and also lined up side by side. Interactions between microcolonies containing several hundred or more bacteria were visible several hours later. Control of cell morphogenesis at later stages of microcolony development was strain specific. These results show that E. coli K-12 cells respond to each other and adjust their cellular morphogenesis to form multicellular groups as they proliferate on agar.     

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique

The growth patterns of microcolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0.89), pseudomonads (r = 0.93) and staphylococci (r = 0.92) after incubation at 30 degrees C for 3 or 6 h (staphylococci) at contamination levels of above 10(3) bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.     

Development and dynamics of Pseudomonas sp. biofilms

Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.     

Electrochemical polarization-induced changes in the growth of individual cells and biofilms of Pseudomonas fluorescens (ATCC 17552)

The effect of surface electrochemical polarization on the growth of cells of Pseudomonas fluorescens (ATCC 17552) on gold electrodes has been examined. Potentials positive or negative to the potential of zero charge (PZC) of gold were applied, and these resulted in changes in cell morphology, size at cell division, time to division, and biofilm structure. At -0.2 V (Ag/AgCl-3 M NaCl), cells elongated at a rate of up to 0.19 microm min(-1), rendering daughter cells that reached up to 3.8 microm immediately after division. The doubling time for the entire population, estimated from the increment in the fraction of surface covered by bacteria, was 82 +/- 7 min. Eight-hour-old biofilms at -0.2 V were composed of large cells distributed in expanded mushroom-like microcolonies that protruded several micrometers in the solution. A different behavior was observed under positive polarization. At an applied potential of 0.5 V, the doubling time of the population was 103 +/- 8 min, cells elongated at a lower rate (up to 0.08 microm min(-1)), rendering shorter daughters (2.5 +/- 0.5 microm) after division, although the duplication times were virtually the same at all potentials. Biofilms grown under this positive potential were composed of short cells distributed in a large number of compact microcolonies. These were flatter than those grown at -0.2 V or at the PZC and were pyramidal in shape. Polarization effects on cell growth and biofilm structure resembled those previously reported as produced by changes in the nutritional level of the culture medium.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.     

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms

A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.     

The relationship between cell size and viability of soil bacteria

The number of bacterial cells in soil that form colonies on nutrient agar represent a small fraction of the direct microscopic counts (DMC). The colony-forming cells have larger cell dimensions than the very small (dwarf) cells which represent the majority of the DMC. This may indicate that the dwarf cells are species unable to form visible colonies on agar, or that they swell to normal dimensions when growing. Indigenous bacterial cells were separated from soil by density gradient centrifugation and fractionated according to diameter by filtration through polycarbonate filters. Each filtrate was studied with respect to DMC, cell dimensions, colony-forming cells (visible colonies and microcolonies), and cell dimensions during growth on the agar. The calculated average percent viability was only 0.2% for cells with diameters below 0.4m, about 10% for cells with diameters between 0.4 and 0.6m, and 30–40% for cells with diameters above 0.6m. Only 10–20% of the viable cells with diameters <0.4m increased their diameter to >0.4m prior to growth. Thus, size change during starvation and growth cycles did not explain the high numbers of dwarf cells observed by microscopy. The results show that despite the relatively low number of colony-forming bacteria in soil, the species that form colonies may be fairly representative for the medium size and large cells, which constitute a major part of the bacterial biovolume. Thus plate counting could be a useful method to count and isolate the bacteria accounting for much of the biovolume in soil. The origin of the dwarf cells is still unclear, but the low number of small cells that increased in size seems to indicate that the majority of these bacterial cells are not small forms of ordinary sized bacteria.     

Surface Attachment Induced Production of Antimicrobial Compounds by Marine Epiphytic Bacteria Using Modified Roller Bottle Cultivation

A modified roller bottle culture method elicited the production of antimicrobial compounds from 2 epibiotic marine bacterial strains, EI-34-6 and II-111-5, isolated from the surface of the marine alga Palmaria palmata. These isolates, tentatively identified as Bacillus species, were grown as a biofilm on the surface of nutrient glycerol ferric agar (NGFA) and marine Columbia glycerol agar (MCGA) on the inside of a rolling bottle. The biofilm was shown to be stable, and the cells were difficult to remove from the agar surface. The culture supernatant exhibited a different antibiotic spectrum when the strains were grown using the agar roller bottle method compared with shake flask cultures or nonagar roller bottle cultures. These results suggest that biofilm formation is an important factor in the production of antimicrobial compounds by these 2 strains, and roller bottle cultivation also allowed production of these compounds to be increased. The methodology used here has the potential to allow increased production of useful secondary metabolites such as antibiotics from marine epibiotic bacteria.     

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique

The growth patterns of macrocolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0·89), pseudomonads (r = 0·93) and staphylococci (r = 0·92) after incubation at 30°C for 3 or 6 h (staphylococci) at contamination levels of above 10³ bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.     

Comparison of microcolony formation between Vibrio sp. strain S141 and a flagellum-negative mutant developing on agar and glass substrata

A flagellum-negative mutant, M8.2, of the marine bacterium Vibrio sp. S141 was produced by transposon mutagenesis. Time-lapse video imaging of surface colonisation behaviour and microcolony formation of S141 compared to M8.2 cells was carried out to investigate the role of the flagellum of Vibrio sp. S141 in microcolony formation on agar and glass substrata. On an agar surface, S141 cells formed a tetrad pattern after the first two cell divisions, during initial surface colonisation. Developed microcolonies consisted of tight circular arrangements of cells with infrequent branching of cells from the main body. In contrast, M8.2 cells did not form tetrad patterns and micro-colonies generally showed enhanced branching and did not develop circular arrangements of cells. On a glass surface under flow conditions, S141 cells displayed several types of movement behaviours at the surface which may have assisted microcolony formation. M8.2 cells appeared unable to develop micro-colonies, but rather displayed a behaviour which enabled them to spread out across the substratum. Laser scanning confocal microscopy revealed S141 mature biofilms consisted of characteristic towers of bacterial growth with scattered troughs. The flagellum-negative M8.2 biofilm did not form such architecture, displaying a homogeneous distribution of cells throughout the biofilm and across the entire substratum. Although not required for attachment to the glass substratum, the flagellum was required for alignment as well as specific movement behaviours by S141 cells.     

New Technique for Mass Assays of Physiological Characteristics of Unicellular Algae

An apparatus is described and illustrated which enables an investigator to inoculate simultaneously a large number of unicellular algal suspensions onto an agar surface. These inoculations may be repeated an indefinite number of times with a very small amount of inoculum. Good results have been obtained from tests in which large numbers of algae were placed onto selective media containing various amounts of many antibiotics, growth factors, sugars, etc.     

Protective effects of agar during immobilization of strains capable to degrade aromatic compounds]

A study of utilization of toxic monoaromatic compounds by microbial cells incorporated into agar matrix revealed positive effects of immobilization on the rate and completeness of biodegradation. This increase in the degrading activity is probably due to a strong protective effect of the polysaccharide carrier. The protective properties of agar are not due to sorptional and diffusional processes in the matrix. These properties were shown to be associated with the formation of a specific extracellular membrane around microcolonies.     

An electron microscopic study of the effect of clindamycin on adherence of Staphylococcus aureus to bone surfaces

Discs of rabbit tibia, 5 mm thick, were utilized to study the adherence of Staphylococcus aureus to the bone surface in the presence and absence of clindamycin. Bacteria were grown in broth media containing the bone slices and varying concentrations of clindamycin. In the absence of the antibiotic, S. aureus adhered extensively to bone surfaces and formed large microcolonies which were surrounded by an amorphous matrix. In the presence of 0.025 micrograms/ml of clindamycin (0.1 MIC), S. aureus adhered less to bone surfaces, forming smaller and fewer microcolonies. In the presence of 0.0625 micrograms/ml of clindamycin (0.25 MIC), S. aureus adhered to the bone surfaces only sparsely, forming small microcolonies with very little matrix holding them together, and leaving very large areas of the bone surface uncolonized. In the presence of 0.125 micrograms/ml of clindamycin (0.5 MIC), bone surfaces were basically clean, with only one or two cells (no microcolonies) found in crevices and indentations of the bone surface. In the presence of 0.25 micrograms/ml (1 MIC) no bacteria adhered to the bone surfaces.