Measurement of Ice Nucleation-Active Bacteria on Plants and in Precipitation by Quantitative PCR

Ice nucleation-active (INA) bacteria may function as high-temperature ice-nucleating particles (INP) in clouds, but their effective contribution to atmospheric processes, i.e., their potential to trigger glaciation and precipitation, remains uncertain. We know little about their abundance on natural vegetation, factors that trigger their release, or persistence of their ice nucleation activity once airborne. To facilitate these investigations, we developed two quantitative PCR (qPCR) tests of the ina gene to directly count INA bacteria in environmental samples. Each of two primer pairs amplified most alleles of the ina gene and, taken together, they should amplify all known alleles. To aid primer design, we collected many new INA isolates. Alignment of their partial ina sequences revealed new and deeply branching clades, including sequences from Pseudomonas syringae pv. atropurpurea, Ps. viridiflava, Pantoea agglomerans, Xanthomonas campestris, and possibly Ps. putida, Ps. auricularis, and Ps. poae. qPCR of leaf washings recorded ∼10⁸ina genes g⁻¹ fresh weight of foliage on cereals and 10⁵ to 10⁷ g⁻¹ on broadleaf crops. Much lower populations were found on most naturally occurring vegetation. In fresh snow, ina genes from various INA bacteria were detected in about half the samples but at abundances that could have accounted for only a minor proportion of INP at −10°C (assuming one ina gene per INA bacterium). Despite this, an apparent biological source contributed an average of ∼85% of INP active at −10°C in snow samples. In contrast, a thunderstorm hail sample contained 0.3 INA bacteria per INP active at −10°C, suggesting a significant contribution to this sample.     

Toxicity of Smoke to Epiphytic Ice Nucleation-Active Bacteria

Wheat straw smoke aerosols and liquid smoke condensates reduced significantly both the viability and the ice-nucleating activity of Pseudomonas syringae pv. syringae and Erwinia herbicola in vitro and on leaf surfaces in vivo. Highly significant reductions in numbers of bacterial ice nuclei on the surface of both corn and almond were observed after exposure to smoke aerosols. At −5°C, frost injury to corn seedlings colonized by ice nucleation-active bacteria was reduced after exposure to smoke aerosols. Effects on −9°C ice nuclei, although significant, were less than on ice nuclei active at −5°C. These results suggest that smoke from wildfires or smudge pots may reduce plant frost susceptibility and sources of ice nuclei important in other natural processes under some conditions.     

Distribution, Population Dynamics, and Characteristics of Ice Nucleation-Active Bacteria in Deciduous Fruit Tree Orchards

Deciduous fruit tree orchards located in the Pacific Northwest were surveyed over a 3-year period for the presence of ice nucleation-active (INA) bacteria. In the Yakima Valley, only about 30% of the fruit tree orchards contained INA bacteria (median population ca. 3 × 10² CFU/g [fresh weight]) in contrast to nearly 75% of the orchards in the Hood River Valley (median population ca. 5 × 10³ CFU/g [fresh weight]). These INA populations ranged from less than 10 to over 10⁶ CFU/g (fresh weight) of blossoms and, in Hood River Valley orchards, generally comprised over 10% of the total bacterial population. Populations of INA bacteria fluctuated during the year with highest levels developing on buds and flowers during the cool, wet spring, followed by a drop in populations during the warmer, drier, summer months and finally a gradual increase in the autumn. The INA bacteria persisted on dormant buds from which they again colonized young developing vegetative tissues. All INA bacteria were identified as Pseudomonas syringae. The frequency of ice nucleation at −5°C for these strains ranged from nearly every cell being INA to less than 1 in 10⁷ cells. The median frequency of ice nucleation at −5°C was 10⁴ cells per ice nucleus. The INA P. syringae strains from individual orchards were diverse with respect to bacteriocin typing and in ice nucleation frequency. The consistent absence of detectable INA bacteria or presence of low populations in most of the orchards surveyed during periods when critical temperatures (i.e., −2 to −5°C) were common indicated a limited role for INA bacteria in frost susceptibility of most Pacific Northwest orchards.     

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at −5 and −12°C. Plants colonized by the non-INA strain were undamaged at −5°C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (−7, −9, and −12°C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At −12°C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.     

Ametryne and Prometryne as Sulfur Sources for Bacteria

Bacteria were isolated that could utilize quantitatively the s-triazine herbicide prometryne [N,N′ -bis(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4-diamine] or ametryne [N-ethyl-N′-(1-methylethyl)-6-(methylthio)-1,3,5-triazine- 2,4-diamine], or both, as a sole source of sulfur for growth. The success of enrichments depended on previous exposure of the soil inoculum to s-triazine herbicides. Deaminoethylametryne [4-(1-methylethyl)amino-6-(methylthio)-1,3,5-triazine-2-(1H)-one], methylsulfonic acid, and sodium sulfate could also be used as sulfur sources. Utilization of a compound was quantified as the growth yield per mole of sulfur supplied. Yields were about 6 kg of protein per mol of sulfur. The product of the desulfuration of an s-triazine was identified as the corresponding hydroxy-derivative. This is the first substantiated report of the utilization of these s-triazines as sulfur sources by bacteria.     

The Presence of Complete but Masked Freezing Nuclei in Various Artificially Constructed Ice Nucleation-Active Proteobacteria

Disparate gamma-subdivision proteobacteria artifically endowed with the same ice gene of enteric origin acquired water-freezing potential at −12°C, but expressed it to varying extents under identical conditions of culture as well as after being subjected to certain post-culture treatments. Varying rates of cell-bound ice nucleus synthesis were probably not the root cause of these observed interspecies differences in nucleation-active cell frequency because potentially functional but masked ice-forming templates were found in the outer cell envelope of even initially inactive individuals taken from physiologically uniform populations of virtually all tested species. We therefore propose that the extent of bacterial ice nucleation generally reflects species-specified extent of ice nucleus sequestration. Received: 4 November 1996 / Accepted: 6 November 1996     

Animal Rennets as Sources of Dairy Lactic Acid Bacteria

The microbial composition of artisan and industrial animal rennet pastes was studied by using both culture-dependent and -independent approaches. Pyrosequencing targeting the 16S rRNA gene allowed to identify 361 operational taxonomic units (OTUs) to the genus/species level. Among lactic acid bacteria (LAB), Streptococcus thermophilus and some lactobacilli, mainly Lactobacillus crispatus and Lactobacillus reuteri, were the most abundant species, with differences among the samples. Twelve groups of microorganisms were targeted by viable plate counts revealing a dominance of mesophilic cocci. All rennets were able to acidify ultrahigh-temperature-processed (UHT) milk as shown by pH and total titratable acidity (TTA). Presumptive LAB isolated at the highest dilutions of acidified milks were phenotypically characterized, grouped, differentiated at the strain level by randomly amplified polymorphic DNA (RAPD)-PCR analysis, and subjected to 16S rRNA gene sequencing. Only 18 strains were clearly identified at the species level, as Enterococcus casseliflavus, Enterococcus faecium, Enterococcus faecalis, Enterococcus lactis, Lactobacillus delbrueckii, and Streptococcus thermophilus, while the other strains, all belonging to the genus Enterococcus, could not be allotted into any previously described species. The phylogenetic analysis showed that these strains might represent different unknown species. All strains were evaluated for their dairy technological performances. All isolates produced diacetyl, and 10 of them produced a rapid pH drop in milk, but only 3 isolates were also autolytic. This work showed that animal rennet pastes can be sources of LAB, mainly enterococci, that might contribute to the microbial diversity associated with dairy productions.     

Methanogenic Bacteria, Including an Acid-Tolerant Strain, from Peatlands

Five pure cultures of methanogenic bacteria were isolated from Minnesota peatlands by enrichment culture techniques. One strain, identified as a member of the family Methanobacteriaceae by antigenic fingerprinting, was acid tolerant and able to produce methane at pH 3.1. Growth could not be demonstrated at pH less than 5.3.     

3-Nitrobenzenesulfonate, 3-Aminobenzenesulfonate, and 4-Aminobenzenesulfonate as Sole Carbon Sources for Bacteria

Aerobic, carbon-limited, enrichment cultures containing 3-aminobenzenesulfonate or 3-nitrobenzenesulfonate as the sole source of carbon and energy yielded growth and complete substrate disappearance. Pure cultures of putative pseudomonads were isolated which utilized these compounds quantitatively. Degradation was compared with that of 2- and 4-aminobenzenesulfonate.     

Fate of Ice Nucleation-Active Pseudomonas syringae Strains in Alpine Soils and Waters and in Synthetic Snow Samples

The stability of the ice nucleation activity (INA) and viability of INA Pseudomonas syringae 31a, used as an ice nucleator in the manufacture of synthetic snow, was determined in snow. The viability of P. syringae 1-2b, a rifampin-resistant mutant selected from strain 31a to improve recovery from test samples, was determined in laboratory tests of three alpine soil and water samples from three different sources. Snow samples were exposed to environmental conditions or held in darkness at −20°C. Samples of soil and water were maintained in darkness at 0, 7.5, or 15°C. Parent strain 31a INA decreased significantly (>99.0%) in snow exposed to sunlight and freeze-thaw, while the INA of the cell population in snow held in darkness at −20°C remained essentially unchanged. No viable strain 31a was detected in snow exposed to the environment after 7 days, while the viability of strain 31a in snow held in darkness at −20°C decreased to <3% of the original inoculation at the test conclusion. Mutant strain 1-2b viability was undetectable or had decreased significantly 19 days postinoculation in soil samples held at 0 or 15°C. In contrast, 1-2b viability remained detectable at low levels for the duration of the test in soils held at 7.5°C. The 1-2b population demonstrated a significantly longer half-life in peatlike soil than in the loam soils tested. The rate of decrease in 1-2b viability was essentially the same in the three alpine water samples tested with respect to water temperature and sample location.     

Aggregated and Free Bacteria as Food Sources for Heterotrophic Microflagellates

Laboratory evidence for the selective grazing of aggregated bacteria by a Bodo sp. and free bacteria by a Paraphysomonas sp. is presented. The relevance of different microflagellate feeding mechanisms with respect to prey distribution in the natural environment is discussed.     

Enumeration of Airborne Bacteria in Hospital

An investigation of the “normal” bacterial content of the air of a large general hospital is described. In many different places within five different areas 70 to 200 settle-plate or slit-sampler bacterial counts were carried out. Average counts were most often of the same order as or lower than other published results and were proportional to human activity. The use of the logarithms of the counts showed no advantage, and conventional statistics should be applied with caution in evaluating such studies. Slitsampler and settle-plate counts of all bacteria showed no correlation, whereas those of Staph. aureus were correlated. There is a lack of parallelism between hospital infection and air bacteria counted by current methods, which are, therefore, not suitable for routine use.     

Butyrate-Producing Bacteria,Including Mucin Degraders,from the Swine Intestinal Tract

To identify bacteria with potential for influencing gut health, 980 anaerobes were cultured from the swine intestinal tract and analyzed for butyrate production. Fifteen isolates in the order Clostridiales produced butyrate and had butyryl coenzyme A (CoA):acetate CoA transferase activity. Three of the isolates grew on mucin, suggesting an intimate association with host intestinal mucosa.     

Proteorhodopsin-Bearing Bacteria in Antarctic Sea Ice

Proteorhodopsins (PRs) are widespread bacterial integral membrane proteins that function as light-driven proton pumps. Antarctic sea ice supports a complex community of autotrophic algae, heterotrophic bacteria, viruses, and protists that are an important food source for higher trophic levels in ice-covered regions of the Southern Ocean. Here, we present the first report of PR-bearing bacteria, both dormant and active, in Antarctic sea ice from a series of sites in the Ross Sea using gene-specific primers. Positive PR sequences were generated from genomic DNA at all depths in sea ice, and these sequences aligned with the classes Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. The sequences showed some similarity to previously reported PR sequences, although most of the sequences were generally distinct. Positive PR sequences were also observed from cDNA reverse transcribed from RNA isolated from sea ice samples. This finding indicates that these sequences were generated from metabolically active cells and suggests that the PR gene is functional within sea ice. Both blue-absorbing and green-absorbing forms of PRs were detected, and only a limited number of blue-absorbing forms were found and were in the midsection of the sea ice profile in this study. Questions still remain regarding the protein''s ecological functions, and ultimately, field experiments will be needed to establish the ecological and functional role of PRs in the sea ice ecosystem.Proteorhodopsins (PRs) are retinal binding bacterial integral membrane proteins that function as light-driven proton pumps (9, 10) and belong to the microbial rhodopsin superfamily of proteins (54). Since the first reported PR sequence from members of SAR86 clade marine (class Gammaproteobacteria) in 2000 (9), many other PR-bearing bacteria have been identified in a range of marine habitats (5, 18, 20, 24, 25, 46, 62). In the recent Global Ocean Sampling (GOS) expedition, almost 4,000 PR sequences from 41 distinct surface marine environments were acquired, demonstrating that these PR genes are extremely abundant in the genomes of ocean bacterioplankton (46). In fact, PR-containing bacteria account for 13% of the community in the Mediterranean Sea and Red Sea and 70% of the community in the Sargasso Sea (18, 46, 49, 60). These light-harvesting bacteria are present in three major marine classes of bacteria: the Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. In addition, two distinct PR genes encode pigments with “blue-absorbing” and “green-absorbing” properties, which is achieved by a substitution at a single amino acid position, which thereby functions as a spectral tuning switch (10, 37, 48).Sea ice represents a complex physicochemical environment in polar regions and covers up to 13% of the Earth''s surface (59). Although extreme gradients of temperature, salinity, nutrient availability, and light stratify the ice matrix from the surface to the ice-water interface (41), the sea ice habitat nevertheless supports a diverse microbial community of phytoplankton, Bacteria, Archaea, viruses, and protists that grow in liquid brine channels within the ice (14, 35, 56). This sea ice microbial community (SIMCO) is highly metabolically active despite being unable to avoid the extreme environmental conditions that they experience (39). In fact, very-high-standing stocks of the SIMCO exist in many regions of the Southern Ocean. For example, the concentration of chlorophyll a, a proxy for microalgal biomass, typically reaches 200 mg m⁻² in the Ross Sea, while the concentration of chlorophyll a in the water column below is approximately 2 orders of magnitude less (47), and the percentage of metabolically active bacteria (32% [39]) is significantly higher than the 10% observed for temperate marine systems (36). The SIMCO is thus a major source of biomass in ice-covered regions of the Southern Ocean (59), providing a critical food source for grazing zooplankton (and, consequently, also for higher trophic levels) for much of the year (3, 59). This biomass is of particular importance during the darkness of the polar winter, where the bottom-ice community is the only available food source for juvenile krill. These grazers absolutely rely on the sea ice microbial community to survive, as the water lacks other food sources (6, 28).In the past decade, reports of the widespread occurrence of bacteriochlorophyll and PR pigments in planktonic marine bacteria have challenged the assumption that chlorophyll a is the only principal light-capturing pigment in ocean surface waters. These alternative pigments may in fact play a critical role in light energy harvesting for microbial metabolism in various aquatic ecosystems (5, 10, 25, 40, 49). It has been proposed that energy, rather than nutrient conservation, is important for the regulation of productivity (7). PR-containing phototrophic eubacteria could play a significant role in the energy budget of cells in the photic zone in marine environments (15). PR sequences have been detected in the Southern Ocean (9), but to our knowledge, there have been no reports of PR-bearing bacteria within the sea ice matrix.The majority of the microbial rhodopsin genes found in oceanic samples have been detected by environmental sequencing (30, 46, 48, 60). We have used degenerate PR gene primers (5) in this study to positively identify PR-bearing operational taxonomic units (OTUs) from sea ice. Also, specific bacterial mRNA can now be detected from extracted nucleic acids and used to examine gene expression and, thus, infer metabolic activity (8). With this in mind, we have generated cDNA from RNA extracted from sea ice samples. From these observations, we deduce that PR-bearing bacteria are present in sea ice and may be actively contributing to the ecosystem within this extreme microenvironment.     

Airborne Bacteria in an Urban Environment

Samples were taken at random intervals over a 2-year period from urban air and tested for viable bacteria. The number of bacteria in each sample was determined, and each organism isolated was identified by its morphological and biochemical characteristics. The number of bacteria found ranged from 0.013 to 1.88 organisms per liter of air sampled. Representatives of 19 different genera were found in 21 samples. The most frequently isolated organisms and their percent of occurence were Micrococcus (41%), Staphylococcus (11%), and Aerococcus (8%). The bacteria isolated were correlated with various weather and air pollution parameters using the Pearson product-moment correlation coefficient method. Statistically significant correlations were found between the number of viable bacteria isolated and the concentrations of nitric oxide (−0.45), nitrogen dioxide (+0.43), and suspended particulate pollutants (+0.56). Calculated individually, the total number of Micrococcus, Aerococcus, and Staphylococcus, number of rods, and number of cocci isolated showed negative correlations with nitric oxide and positive correlations with nitrogen dioxide and particulates. Statistically significant positive correlations were found between the total number of rods isolated and the concentration of nitrogen dioxide (+0.54) and the percent relative humidity (+0.43). The other parameters tested, sulfur dioxide, hydrocarbons, and temperature, showed no significant correlations.