Bacterial colonization of particles: growth and interactions

Marine particles in the ocean are exposed to diverse bacterial communities, and colonization and growth of attached bacteria are important processes in the degradation and transformation of the particles. In an earlier study, we showed that the initial colonization of model particles by individual bacterial strains isolated from marine aggregates was a function of attachment and detachment. In the present study, we have investigated how this colonization process was further affected by growth and interspecific interactions among the bacteria. Long-term incubation experiments showed that growth dominated over attachment and detachment after a few hours in controlling the bacterial population density on agar particles. In the absence of grazing mortality, this growth led to an equilibrium population density consistent with the theoretical limit due to oxygen diffusion. Interspecific interaction experiments showed that the presence of some bacterial strains ("residents") on the agar particles either increased or decreased the colonization rate of other strains ("newcomers"). Comparison between an antibiotic-producing strain and its antibiotic-free mutant showed no inhibitory effect on the newcomers due to antibiotic production. On the contrary, hydrolytic activity of the antibiotic-producing strain appeared to benefit the newcomers and enhance their colonization rate. These results show that growth- and species-specific interactions have to be taken into account to adequately describe bacterial colonization of marine particles. Changes in colonization pattern due to such small-scale processes may have profound effects on the transformation and fluxes of particulate matter in the ocean.     

Iron effects on colonization behavior, motility, and enzymatic activity of marine bacteria

Iron availability in the ocean has been shown to affect the growth and production of phytoplankton and free-living bacteria. A large fraction of marine bacteria are specialized in colonizing and living on particles and aggregates, but the effects of iron limitation on these bacteria are not fully known. We conducted laboratory experiments to study the effects of iron availability on particle colonization behavior, motility, and enzymatic activities of 4 strains of marine bacteria. Iron depletion reduced the bacterial particle colonization rate by 1.7%-43.1%, which could be attributed to reduced swimming speeds in 2 of the 4 strains. Protease activity was not affected by iron availability. However, attached bacteria did show higher protease activities than their free counterparts. Our results suggest that iron limitation in the ocean could in some cases reduce bacteria-particle interactions by reducing bacterial motility and colonization rate.     

Ecology and Physics of Bacterial Chemotaxis in the Ocean

Summary: Intuitively, it may seem that from the perspective of an individual bacterium the ocean is a vast, dilute, and largely homogeneous environment. Microbial oceanographers have typically considered the ocean from this point of view. In reality, marine bacteria inhabit a chemical seascape that is highly heterogeneous down to the microscale, owing to ubiquitous nutrient patches, plumes, and gradients. Exudation and excretion of dissolved matter by larger organisms, lysis events, particles, animal surfaces, and fluxes from the sediment-water interface all contribute to create strong and pervasive heterogeneity, where chemotaxis may provide a significant fitness advantage to bacteria. The dynamic nature of the ocean imposes strong selective pressures on bacterial foraging strategies, and many marine bacteria indeed display adaptations that characterize their chemotactic motility as “high performance” compared to that of enteric model organisms. Fast swimming speeds, strongly directional responses, and effective turning and steering strategies ensure that marine bacteria can successfully use chemotaxis to very rapidly respond to chemical gradients in the ocean. These fast responses are advantageous in a broad range of ecological processes, including attaching to particles, exploiting particle plumes, retaining position close to phytoplankton cells, colonizing host animals, and hovering at a preferred height above the sediment-water interface. At larger scales, these responses can impact ocean biogeochemistry by increasing the rates of chemical transformation, influencing the flux of sinking material, and potentially altering the balance of biomass incorporation versus respiration. This review highlights the physical and ecological processes underpinning bacterial motility and chemotaxis in the ocean, describes the current state of knowledge of chemotaxis in marine bacteria, and summarizes our understanding of how these microscale dynamics scale up to affect ecosystem-scale processes in the sea.     

Effect of Motility on Surface Colonization and Reproductive Success of Pseudomonas fluorescens in Dual-Dilution Continuous Culture and Batch Culture Systems

The colonization of glass surfaces by motile and nonmotile strains of Pseudomonas fluorescens was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10⁸ versus 5.57 × 10⁷ cells vial^-1) whether evaluated alone or in cocultures containing motile and nonmotile P. fluorescens. These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile P. fluorescens colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.     

Inhibitory activity of antibiotic-producing marine bacteria against fish pathogens

The activity of antibiotic-producing marine bacteria was assayed against bacterial fish pathogens belonging to the genera Vibrio, Aeromonas, Pasteurella, Edwardsiella, Yersinia and Pseudomonas with the aim of evaluating the possible use of these marine strains for controlling epizootics in aquaculture. Inhibition tests on solid medium showed that, in general, the majority of fish bacteria were strongly sensitive to the marine bacteria. Only two strains ( Edwardsiella tarda and Pseudomonas aeruginosa ), were resistant to all the antibiotic-producing strains. The results of antagonism assays in sea water, however, varied according to the fish pathogens examined. Experiments conducted using cell-free supernatant fluids of marine bacteria demonstrated the involvement of antibiotic substances in the inhibition of fish pathogens.     

Inhibitory activity of antibiotic-producing marine bacteria against fish pathogens

The activity of antibiotic-producing marine bacteria was assayed against bacterial fish pathogens belonging to the genera Vibrio, Aeromonas, Pasteurella, Edwardsiella, Yersinia and Pseudomonas with the aim of evaluating the possible use of these marine strains for controlling epizootics in aquaculture. Inhibition tests on solid medium showed that, in general, the majority of fish bacteria were strongly sensitive to the marine bacteria. Only two strains (Edwardsiella tarda and Pseudomonas aeruginosa), were resistant to all the antibiotic-producing strains. The results of antagonism assays in sea water, however, varied according to the fish pathogens examined. Experiments conducted using cell-free supernatant fluids of marine bacteria demonstrated the involvement of antibiotic substances in the inhibition of fish pathogens.     

Competitive dominance of antibiotic-producing marine bacteria in mixed cultures

Competitive relationships between antibiotic-producing marine bacteria and other non-producers were studied in seawater mixed cultures. Producer strains showed a competitive advantage against non-producers as the latter were inhibited after a short time. Inhibition was also noted in mixed cultures of two producer strains. The inhibitory effect was not observed in a mixed culture with two non-producers, which indicates that an amensalist interaction occurred between populations of antibiotic-producing and non-producing marine bacteria. The results suggest that antibiotics could play an important role in the competitive relationships between marine bacterial populations.     

Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds

A survey of antibiotic-producing bacteria from the microbial flora attached to seaweeds and the study of their antibiotic capacities were carried out. From 5 species of green and brown marine algae, 224 bacterial strains were isolated and tested for antibiotic production. A total of 38 strains displayed antibiotic activity, withEnteromorpha intestinalis being the source of the highest number of producer strains. All epiphytic bacteria with antibiotic activity were assigned to thePseudomonas-Alteromonas group. Antagonism assays among the isolates demonstrated that each producer strain inhibits the growth of the other producers, as well as of some nonproducer strains also isolated from seaweeds. Likewise, an autoinhibitory effect was observed in all antibiotic-producing strains. Antibacterial spectra of all the strains include activity againstStaphylococcus, Alcaligenes, Pseudomonas, Vibrio, Pasteurella, andAchromobacter. A preliminary characterization of the antibiotic substances produced by these epiphytic bacteria demonstrated that they are low molecular weight compounds, thermolabile, and anionic and are not affected by proteolytic enzymes. The role that these inhibitory substances can play in the natural environment is discussed.     

Masking of antibiotic-resistance upon recovery of endophytic bacteria

During studies on internal plant colonization by rhizosphere bacteria and endophytic bacteria over several years, we frequently observed lack of growth of rifampicin-resistant mutants (rif+) on tryptic soy agar amended with rifampicin (RTSA). Following seed treatment of cucumber with 6 species of rif+ rhizosphere bacteria in one experiment, all strains were recoverable on RTSA when external root colonization was monitored. Following trituration of surface-disinfested roots, only one strain grew directly on RTSA; however colonies isolated on tryptic soy agar (TSA) grew within 18 h after transfer to RTSA. We term this temporary loss of the antibiotic-resistant phenotype ‘antibiotic masking’. Antibiotic masking was also observed with isolation of 7 rif+ endophytic bacterial strains from inside stems of cotton and with isolation of mutants of bacterial endophytes resistant to polymyxin B sulfate from cotton plants. Rifampicin-masking was not accounted for in vitro by inhibitory compounds from cotton plant extracts, by bacterial growth on low nutrient agar, or by competition with other bacteria. Collectively, these results suggest that expression of antibiotic-resistance may be altered in planta, although causes for this antibiotic-masking remain to be elucidated, methods for quantifying internal plant colonization by rif+ bacteria should account for this possibility. ei]Section editor: R O D Dixon     

Competitive dominance of antibiotic-producing marine bacteria in mixed cultures

M.L. LEMOS. C.P. DOPAZO, ALICIA E. TORANZO AND J. L. BARJA. 1991. Competitive relationships between antibiotic-producing marine bacteria and other non-producers were studied in seawater mixed cultures. Producer strains showed a competitive advantage against non-producers as the latter were inhibited after a short time. Inhibition was also noted in mixed cultures of two producer strains. The inhibitory effect was not observed in a mixed culture with two non-producers, which indicates that an amensalist interaction occurred between populations of antibiotic-producing and non-producing marine bacteria. The results suggest that antibiotics could play an important role in the competitive relationships between marine bacterial populations.     

Antagonistic Interactions among Marine Bacteria Impede the Proliferation of Vibrio cholerae

Changes in global climate have raised concerns about the emergence and resurgence of infectious diseases. Vibrio cholerae is a reemerging pathogen that proliferates and is transported on marine particles. Patterns of cholera outbreaks correlate with sea surface temperature increases, but the underlying mechanisms for rapid proliferation of V. cholerae during ocean warming events have yet to be fully elucidated. In this study, we tested the hypothesis that autochthonous marine bacteria impede the spread of V. cholerae in the marine environment. It was found that some marine bacteria are capable of inhibiting the growth of V. cholerae on surfaces and that bacterial isolates derived from pelagic particles show a greater frequency of V. cholerae inhibition than free-living bacteria. Vibrio cholerae was less susceptible to antagonism at higher temperatures, such as those measured during El Niño-Southern Oscilliation and monsoonal events. Using a model system employing green fluorescent protein-labeled bacteria, we found that marine bacteria can directly inhibit V. cholerae colonization of particles. The mechanism of inhibition in our model system was linked to the biosynthesis of andrimid, an antibacterial agent. Antibiotic production by the model antagonistic strain decreased at higher temperatures, thereby explaining the increased competitiveness of V. cholerae under warmer conditions. These findings suggest that bacterium-bacterium antagonism is a contributing mechanism in regulating the proliferation of V. cholerae on marine particles.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

Antagonistic interactions among marine bacteria impede the proliferation of Vibrio cholerae

Changes in global climate have raised concerns about the emergence and resurgence of infectious diseases. Vibrio cholerae is a reemerging pathogen that proliferates and is transported on marine particles. Patterns of cholera outbreaks correlate with sea surface temperature increases, but the underlying mechanisms for rapid proliferation of V. cholerae during ocean warming events have yet to be fully elucidated. In this study, we tested the hypothesis that autochthonous marine bacteria impede the spread of V. cholerae in the marine environment. It was found that some marine bacteria are capable of inhibiting the growth of V. cholerae on surfaces and that bacterial isolates derived from pelagic particles show a greater frequency of V. cholerae inhibition than free-living bacteria. Vibrio cholerae was less susceptible to antagonism at higher temperatures, such as those measured during El Ni?o-Southern Oscilliation and monsoonal events. Using a model system employing green fluorescent protein-labeled bacteria, we found that marine bacteria can directly inhibit V. cholerae colonization of particles. The mechanism of inhibition in our model system was linked to the biosynthesis of andrimid, an antibacterial agent. Antibiotic production by the model antagonistic strain decreased at higher temperatures, thereby explaining the increased competitiveness of V. cholerae under warmer conditions. These findings suggest that bacterium-bacterium antagonism is a contributing mechanism in regulating the proliferation of V. cholerae on marine particles.     

Comparison of cell-specific activity between free-living and attached bacteria using isolates and natural assemblages

Marine snow aggregates are microbial hotspots that support high bacterial abundance and activities. We conducted laboratory experiments to compare cell-specific bacterial protein production (BPP) and protease activity between free-living and attached bacteria. Natural bacterial assemblages attached to model aggregates (agar spheres) had threefold higher BPP and two orders of magnitude higher protease activity than their free-living counterpart. These observations could be explained by preferential colonization of the agar spheres by bacteria with inherently higher metabolic activity and/or individual bacteria increasing their metabolism upon attachment to surfaces. In subsequent experiments, we used four strains of marine snow bacteria isolates to test the hypothesis that bacteria could up- and down-regulate their metabolism while on and off an aggregate. The protease activity of attached bacteria was 10-20 times higher than that of free-living bacteria, indicating that the individual strains could increase their protease activity within a short time (2 h) upon attachment to surfaces. Agar spheres with embedded diatom cells were colonized faster than plain agar spheres and the attached bacteria were clustered around the agar-embedded diatom cells, indicating a chemosensing response. Increased protease activity and BPP allow attached bacteria to quickly exploit aggregate resources upon attachment, which may accelerate remineralization of marine snow and reduce the downward carbon fluxes.     

Mechanisms and Rates of Bacterial Colonization of Sinking Aggregates

Quantifying the rate at which bacteria colonize aggregates is a key to understanding microbial turnover of aggregates. We used encounter models based on random walk and advection-diffusion considerations to predict colonization rates from the bacteria's motility patterns (swimming speed, tumbling frequency, and turn angles) and the hydrodynamic environment (stationary versus sinking aggregates). We then experimentally tested the models with 10 strains of bacteria isolated from marine particles: two strains were nonmotile; the rest were swimming at 20 to 60 μm s⁻¹ with different tumbling frequency (0 to 2 s⁻¹). The rates at which these bacteria colonized artificial aggregates (stationary and sinking) largely agreed with model predictions. We report several findings. (i) Motile bacteria rapidly colonize aggregates, whereas nonmotile bacteria do not. (ii) Flow enhances colonization rates. (iii) Tumbling strains colonize aggregates enriched with organic substrates faster than unenriched aggregates, while a nontumbling strain did not. (iv) Once on the aggregates, the bacteria may detach and typical residence time is about 3 h. Thus, there is a rapid exchange between attached and free bacteria. (v) With the motility patterns observed, freely swimming bacteria will encounter an aggregate in <1 day at typical upper-ocean aggregate concentrations. This is faster than even starving bacteria burn up their reserves, and bacteria may therefore rely solely on aggregates for food. (vi) The net result of colonization and detachment leads to a predicted equilibrium abundance of attached bacteria as a function of aggregate size, which is markedly different from field observations. This discrepancy suggests that inter- and intraspecific interactions among bacteria and between bacteria and their predators may be more important than colonization in governing the population dynamics of bacteria on natural aggregates.     

Analysis of Bacterial Detachment from Substratum Surfaces by the Passage of Air-Liquid Interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 × 10⁶ cells cm⁻² was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s⁻¹), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as “bacterial retention” rather than “bacterial adhesion”.     

Marine Sediment Bacteria Harbor Antibiotic Resistance Genes Highly Similar to Those Found in Human Pathogens

The ocean is a natural habitat for antibiotic-producing bacteria, and marine aquaculture introduces antibiotics into the ocean to treat infections and improve aquaculture production. Studies have shown that the ocean is an important reservoir of antibiotic resistance genes. However, there is a lack of understanding and knowledge about the clinical importance of the ocean resistome. We investigated the relationship between the ocean bacterial resistome and pathogenic resistome. We applied high-throughput sequencing and metagenomic analyses to explore the resistance genes in bacterial plasmids from marine sediments. Numerous putative resistance determinants were detected among the resistance genes in the sediment bacteria. We also found that several contigs shared high identity with transposons or plasmids from human pathogens, indicating that the sediment bacteria recently contributed or acquired resistance genes from pathogens. Marine sediment bacteria could play an important role in the global exchange of antibiotic resistance.     

Role of alginate lyase in cell detachment of Pseudomonas aeruginosa.

The exopolysaccharide alginate of Pseudomonas aeruginosa was shown to be important in determining the degree of cell detachment from an agar surface. Nonmucoid strain 8822 gave rise to 50-fold more sloughed cells than mucoid strains 8821 and 8830. Alginate anchors the bacteria to the agar surface, thereby influencing the extent of detachment. The role of the P. aeruginosa alginate lyase in the process of cell sloughing was investigated. Increased expression of the alginate lyase in mucoid strain 8830 led to alginate degradation and increased cell detachment. Similar effects were seen both when the alginate lyase was induced at the initial stage of cell inoculation and when it was induced at a later stage of growth. It appears that high-molecular-weight alginate polymers are required to efficiently retain the bacteria within the growth film. When expressed from a regulated promoter, the alginate lyase can induce enhanced sloughing of cells because of degradation of the alginate. This suggests a possible role for the lyase in the development of bacterial growth films.     

Phenotypic Signatures Arising from Unbalanced Bacterial Growth

Fluctuations in the growth rate of a bacterial culture during unbalanced growth are generally considered undesirable in quantitative studies of bacterial physiology. Under well-controlled experimental conditions, however, these fluctuations are not random but instead reflect the interplay between intra-cellular networks underlying bacterial growth and the growth environment. Therefore, these fluctuations could be considered quantitative phenotypes of the bacteria under a specific growth condition. Here, we present a method to identify “phenotypic signatures” by time-frequency analysis of unbalanced growth curves measured with high temporal resolution. The signatures are then applied to differentiate amongst different bacterial strains or the same strain under different growth conditions, and to identify the essential architecture of the gene network underlying the observed growth dynamics. Our method has implications for both basic understanding of bacterial physiology and for the classification of bacterial strains.     

Significance of Size and Nucleic Acid Content Heterogeneity as Measured by Flow Cytometry in Natural Planktonic Bacteria

Total bacterial abundances estimated with different epifluorescence microscopy methods (4′,6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, “live” bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and “dead” bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of “live” cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.