Pink-pigmented Facultative Methylotrophs Research Articles

Analysis of Pink Pigmented Facultative Methylotroph Bacteria from Human Environments

The formation of pink biofilm in wet places are usually correlated with chlorine-resistant pink pigmented facultative methylotrophs (PPFM). In this study we investigated the presence of PPFM bacteria through bacterial isolation and detection of mxaF gene from wet places of human-made environments. A total of eighteen PPFM bacterial isolates were recovered from the formation of biofilm bacterial of four test places such as washstands, bathrooms, and potable water supplies. Confirmation of the isolates through biochemical analysis were done using catalase, oxidase and urease tests. Chlorine-resistance-activity was assayed for all of the isolates. Antibiotic resistance were examined for ampicillin (25 μg), tetracycline (30 μg), kanamycin (30 μg), trimethoprim (1.25 μg), and streptomycin (10 μg) using the agar diffusion method. Genomic DNA was subjected to PCR analysis with primers corresponding to the 5’- and 3’- end conserved segments of the mxaF gene. PCR amplification followed by DNA sequencing of 16S rRNA gene were done for some isolates. We recovered 18 isolates of PPFM bacteria. Biochemical analysis indicated that the isolates were positive for catalase, oxidase, and urease activities. Chlorine-resistance-analysis showed the majority of the isolates were resistant to chlorine. Antibiotic resistance assays showed all of the isolates exhibited resistance to trimethoprim but were sensitive to streptomycin, kanamycin, and tetracycline but were variably resistant to ampicilin. PCR detection using specific primers for the mxaF gene gave a positive result for all of the isolates. DNA sequencing of the 16S rRNA gene of two isolates showed that isolate WD10 had a 98% similarity with the mxaF gene from Methylobacterium lusitanum strain MP2 and isolate WK2 had a 98% similarity to the mxaF gene from Afipia felis strain RD1. The formation of pink biofilm of four wet areas in this study were correlated with the presence of chlorine-resistant PPFM bacteria and we confirmed with the presence of the mxaF gene in all of the isolates. This finding needs to be widely publicized since some PPFM bacteria were known as opportunistic pathogens.

Analysis of Pink Pigmented Facultative Methylotroph Bacteria from Human Environments

The formation of pink biofilm in wet places are usually correlated with chlorine-resistant pink pigmented facultative methylotrophs (PPFM). In this study we investigated the presence of PPFM bacteria through bacterial isolation and detection of mxaF gene from wet places of human-made environments. A total of eighteen PPFM bacterial isolates were recovered from the formation of biofilm bacterial of four test places such as washstands, bathrooms, and potable water supplies. Confirmation of the isolates through biochemical analysis were done using catalase, oxidase and urease tests. Chlorine-resistance-activity was assayed for all of the isolates. Antibiotic resistance were examined for ampicillin (25 μg), tetracycline (30 μg), kanamycin (30 μg), trimethoprim (1.25 μg), and streptomycin (10 μg) using the agar diffusion method. Genomic DNA was subjected to PCR analysis with primers corresponding to the 5’- and 3’- end conserved segments of the mxaF gene. PCR amplification followed by DNA sequencing of 16S rRNA gene were done for some isolates. We recovered 18 isolates of PPFM bacteria. Biochemical analysis indicated that the isolates were positive for catalase, oxidase, and urease activities. Chlorine-resistance-analysis showed the majority of the isolates were resistant to chlorine. Antibiotic resistance assays showed all of the isolates exhibited resistance to trimethoprim but were sensitive to streptomycin, kanamycin, and tetracycline but were variably resistant to ampicilin. PCR detection using specific primers for the mxaF gene gave a positive result for all of the isolates. DNA sequencing of the 16S rRNA gene of two isolates showed that isolate WD10 had a 98% similarity with the mxaF gene from Methylobacterium lusitanum strain MP2 and isolate WK2 had a 98% similarity to the mxaF gene from Afipia felis strain RD1. The formation of pink biofilm of four wet areas in this study were correlated with the presence of chlorine-resistant PPFM bacteria and we confirmed with the presence of the mxaF gene in all of the isolates. This finding needs to be widely publicized since some PPFM bacteria were known as opportunistic pathogens.

Impact of transgenic Bt-cotton on the diversity of pink-pigmented facultative methylotrophs

Pink-pigmented facultative methylotrophs (PPFMs) are one of the beneficial proteobacteria commonly found in phyllosphere, rhizosphere and as endophytes in cotton. To assess the impact of transgenic Bt-cotton on changes in the diversity and community profile of PPFMs by comparing with its non-transgenic cotton, a polyphasic approach including differential carbon-substrate utilization profiling and DNA fingerprinting techniques like ARDRA, RISA, BOX-PCR and ERIC-PCR were studied. PPFMs from phyllosphere, rhizoplane and internal tissues of the stem of both Bt-cotton and non-Bt-cotton were isolated and analysed in this study. All the results suggested that the diversity richness of PPFMs present in the phyllosphere, rhizoplane and internal tissues did not differ between Bt- and non-Bt-cotton. In this study, there was no evidence to indicate any adverse effects of Bt-cotton on the diversity of plant-associated methylobacteria.

Genetic and metabolic diversity of pink-pigmented facultative methylotrophs in phyllosphere of tropical plants

Genetic and metabolic diversity of pink-pigmented facultative methylotrophs in phyllosphere of tropical plants

Genetic and metabolic diversity of pink-pigmented facultative methylotrophs in phyllosphere of tropical plants.

Diversity of Pink-Pigmented Facultative Methylotrophs (PPFMs) in phyllosphere of cotton, maize and sunflower was determined based on differential carbon-substrate utilization profile and Random Amplified Polymorphic DNA data. Results indicate that six diversified groups of PPFMs are found in these crops. Sunflower and maize phyllosphere harbor four different groups of methylobacteria while cotton has only two groups.

Molecular interaction between Methylobacterium extorquens and seedlings: growth promotion, methanol consumption, and localization of the methanol emission site

Four Methylobacterium extorquens strains were isolated from strawberry (Fragaria x ananassa cv. Elsanta) leaves, and one strain, called ME4, was tested for its ability to promote the growth of various plant seedlings. Seedling weight and shoot length of Nicotiana tabacum, Lycopersicon esculentum, Sinapis alba, and Fragaria vesca increased significantly in the presence of the pink-pigmented facultative methylotroph (PPFM), but the germination behaviour of seeds from six other plants was not affected. The cell-free supernatant of the bacterial culture stimulated germination, suggesting the production of a growth-promoting agent by the methylotroph. Methanol emitted from N. tabacum seedlings, as determined by proton-transfer-reaction mass spectrometry (PTR-MS), ranged from 0.4 to 0.7 ppbv (parts per billion by volume), while significantly lower levels (0.005 to 0.01 ppbv) of the volatile alcohol were measured when the seedlings were co-cultivated with M. extorquens ME4, demonstrating the consumption of the gaseous methanol by the bacteria. Additionally, by using cells of the methylotrophic yeast Pichia pastoris transformed with the pPICHS/GFP vector harbouring a methanol-sensitive promoter in combination with the green fluorescence protein (GFP) reporter gene, stomata were identified as the main source of the methanol emission on tobacco cotyledons. Methylobacterium extorquens strains can nourish themselves using the methanol released by the stomata and release an agent promoting the growth of the seedlings of some crop plants.

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at alpha concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.

Characterization of Ni-tolerant methylobacteria associated with the hyperaccumulating plant Thlaspi goesingense and description of Methylobacterium goesingense sp. nov.

Various pink-pigmented facultative methylotrophic (PPFM) bacteria (strains iEII3, iEIV1, iEI6, iEII1, iEIII3 iEIII4, iEIII5, iRII1, iRII2, iRIII1, iRIV1 and iRIV2) were obtained from the rhizosphere and endosphere of hyperaccumulating plant Thlaspi goesingense grown in Redschlag, Austria [R. Idris, R. Trifonova, M. Puschenreiter, W.W. Wenzel, A. Sessitsch, Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense, Appl. Environ. Microbiol. 70 (2004) 2667–2677]. Due to their unexpected diversity, abundance and nickel tolerance they were further characterized by detailed 16S rRNA gene analysis, DNA–DNA hybridization, fatty acid analysis, heavy metal tolerance, screening for known Ni resistance genes and phenotypic analysis. These strains were found to exhibit different multiple heavy metal resistance characteristics to Ni, Cd, Co, Zn and Cr. On the basis of their physiological and genotypic properties, strains could be grouped with Methylobacterium extorquens and M. mesophilicum. One endophyte, strain iEII3, was found to belong to a novel species for which the name M. goesingense is proposed.

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain ( N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

A new insight into foliar applied methanol influencing phylloplane methylotrophic dynamics and growth promotion of cotton ( Gossypium hirsutum L.) and sugarcane ( Saccharum officinarum L.)

Foliar applied methanol has been purported to enhance growth and yield of cotton and sugarcane possibly by stimulation of plant hormone production mediated by pink-pigmented facultative methylotrophic (PPFMs) bacteria. In vitro studies were performed on the relations between leaf methanol and pectin methylesterase (PME: EC 3.1.1.11) in young and old leaves of cotton and sugarcane. Results of field trials and pot culture studies of cotton showed that application of 30% methanol or PPFMs as foliar spray significantly increased plant height, plant dry weight, leaf area, boll number, and boll dry weight, leading to an increase of seed cotton yield (SCY) over control. Foliar application of PPFMs increased plant height and specific leaf area of sugarcane and led to a cane yield increase of 9.8% over control. The overall PPFMs population in the phyllosphere of cotton remained higher than sugarcane. Applications of methanol or PPFMs increased the total cytokinins in cotton and sugarcane. The methanol emission process, regulated by PME activity that catalyzes demethoxylation of pectins, could trigger PPFMs population on the leaf surface and subsequent cytokinin production in plants, and might play a role in plant growth promotion. In our study, the foliar applications of methanol or PPFMs increased the PPFM populations and cytokinin production resulting in increased yield in cotton and sugarcane.

Metabolism of the Aliphatic Nitramine 4-Nitro-2,4-Diazabutanal by Methylobacterium sp. Strain JS178

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C2H5N3O3) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various Rhodococcus spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus Methylobacterium. The strain, named Methylobacterium sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N2O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO2-), or ammonium (NH4+) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [14C]NDAB produced in situ from [14C]RDX by Rhodococcus sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX.

Indole-3-acetic acid production by pink-pigmented facultative methylotrophic bacteria

In examining the presence of indole-3-acetic acid (IAA) in supernatants of pink-pigmented facultativemethylotrophic (PPFMs) bacterial cultures, three out of the 16 isolates tested showed a positive reaction ina colorimetric assay. The presence was further unambiguously con?rmed by high-performance liquidchromatography in combination with NMR. The IAA production was signi?cantly stimulated byL-tryptophan. These results prove that PPFM bacteria are able to produce the plant hormone IAA.

Plants in the pink: cytokinin production by methylobacterium.

Bacteria of the genus Methylobacterium are well-studied examples of facultative methylotrophs. These bacteria are classified as α-proteobacteria and are capable of growth on methanol and methylamine as well as on a variety of C2, C3, and C4 compounds (8). Methylobacterium strains are commonly found in soils, as well as on the surfaces of leaves of a wide variety of plants (1). Because of their distinctive pink pigmentation, they are sometimes referred to as PPFMs (pink-pigmented facultative methylotrophs). Evidence exists that these bacteria utilize methanol emitted by the stomata of plants (9), but the details of their relationship to plants has been unclear. Are Methylobacterium strains commensal bacteria, or do they communicate with plants in a more intimate relationship? A number of reports suggest that Methylobacterium strains are more than passive passengers on plant leaves. For instance, they have been shown in some cases to stimulate seed germination and plant development, possibly by production of phytohormones (2, 3, 4), and it has been reported that one strain produces the cytokinin zeatin (5) while others have been reported to produce indole acetic acid (6). In addition, Methylobacterium strains have been suggested to contribute to the flavor of strawberries (12) and have been localized as endosymbionts within cells of the buds of Scotch pine (Pinus sylvestris) (10). One nonpigmented Methylobacterium strain has been shown to form a root-nodulating nitrogen-fixing symbiosis with a legume (11). A partial genome sequence of Methylobacterium extorquens AM1 is available (http://pedant.mips.biochem.mpg.de/), which reveals a number of open reading frames with significant identity to genes involved in plant association in rhizobia and Agrobacterium (M. Lidstrom, unpublished results). Therefore, at this point numerous indications exist that these PPFMs interact with plants, but the biochemical and genetic details of those interactions have remained elusive. In this issue, Koenig et al. (7) present a set of studies that represents an important step in understanding the molecular basis of Methylobacterium-plant interactions. They show that four different Methylobacterium strains representing leaf isolates and a type M. extorquens strain all produce the cytokinin trans-zeatin at low levels in pure culture and excrete it into the culture medium. Biochemical and genetic evidence is presented suggesting that the trans-zeatin is not synthesized de novo but instead is derived from tRNA. This result is surprising, since so far the tRNA-derived zeatin from bacteria and plants has been restricted to the cis isomer. Is the low-level production of trans-zeatin observed in these strains sufficient to have an effect on plants? Surprisingly, the Methylobacterium-specific stimulation of seed germination that has been described in the past (3, 4) was unaffected in a mutant that is incapable of producing trans-zeatin. Thus, the answer to this question is still outstanding, as is the source of the stimulation effect itself. However, the results presented by Koenig et al. provide mechanistic evidence for cytokinin production by Methylobacterium strains, leading the way for future studies of the Methylobacterium-plant relationship. In addition, this evidence that a commensal bacterium produces a phytohormone via a previously unknown route provides new insights into the role of commensal plant bacteria in general.

Bio inoculants for enhancing the productivity and nutrient uptake of winter irrigated cotton (Gossypium hirsutum) under graded levels of nitrogen and phosphatic fertilizers

Field experiments were conducted during winter (August February) seasons of 2003-04 and 2004-05 under irrigated condition at Coimbatore to study the usefulness of bio-inoculants in reducing the inorganic nitrogen and phosphatic fertilizers for cotton. Four graded levels of N and P ( 0,50,75 and 100 % recommended dose (RDNP) and four bio inoculants viz ., Azospir illum from, Haryana, Tamil Nadu and Surat were combined with Phosphorus Solubilizing Bacteria (PSB) + Pink Pigmented Facultative Methylotrophs (PPFM) along with uninoculated control. Cotton LRA 5166 responded significantly to graded levels of N and P and bio-inoculants. Application of 75% RDNP along with Azospirillum (HAU) + PSB + PPFM yielded 2.91 tonne/ha seed cotton with higher net return (Rs 40,553) and net B:C ratio value of 1.94 while, application of 100% RDNP without bio-inoculants yielded only 2.54 tonne/ha. Judicious combination of bio-inoculants along with moderate use of chemical fertilizers is recommended for cotton.

Facultative and obligate aerobic methylobacteria synthesize cytokinins

The presence and expression of genes controlling the synthesis and secretion of cytokinins by the pink-pigmented facultative methylotroph Methylobacterium mesophilicum VKM B-2143 with the serine pathway and nonpigmented obligate methylotroph Methylovorus mays VKM B-2221 with the ribulose monophosphate pathway of C1 metabolism were shown using the polymerase chain reaction (PCR) and reverse transcription-PCR methods. The presence of the corresponding mRNA in M. mesophilicum cells grown on methanol or succinate suggests that the expression of these genes is constitutive. The cytokinin activity of culture liquid and its fractions was determined by a biotest with Amarantus caudatus L. seedlings. Using enzyme-linked immunosorbent analysis, we detected zeatin (riboside) in the culture liquid of both bacteria studied. The data obtained show that the aerobic methylobacteria are phytosymbionts that are able to utilize the single- and polycarbon compounds secreted by symbiotic plants and to synthesize cytokinins.

Isolation and characterization of two new methanesulfonic acid-degrading bacterial isolates from a Portuguese soil sample.

Two novel bacterial strains that can utilize methanesulfonic acid as a source of carbon and energy were isolated from a soil sample collected in northern Portugal. Morphological, physiological, biochemical and molecular biological characterization of the two isolates indicate that strain P1 is a pink-pigmented facultative methylotroph belonging to the genus Methylobacterium, while strain P2 is a restricted methylotroph belonging to the genus Hyphomicrobium. Both strains are strictly aerobic, degrade methanesulfonate, and release small quantities of sulfite into the medium. Growth on methanesulfonate induces a specific polypeptide profile in each strain. This, together with the positive hybridization to a DNA probe that carries the msm genes of Methylosulfonomonas methylovora strain M2, strongly endorses the contention that a methanesulfonic acid monooxygenase related to that found in the previously known methanesulfonate-utilizing bacteria is present in strains P1 and P2. The isolation of bacteria containing conserved msm genes from diverse environments and geographical locations supports the hypothesis that a common enzyme may be globally responsible for the oxidation of methanesulfonate by natural methylotrophic communities.

Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation

Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C 1 metabolism with biochemical and molecular biological techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.

Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation.

Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C1 metabolism with biochemical and molecular biological techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.

A novel pink-pigmented facultative methylotroph, Methylobacterium thiocyanatum sp. nov., capable of growth on thiocyanate or cyanate as sole nitrogen sources

The isolation and properties of a novel species of pink-pigmented methylotroph, Methylobacterium thiocyanatum, are described. This organism satisfied all the morphological, biochemical, and growth-substrate criteria to be placed in the genus Methylobacterium. Sequencing of the gene encoding its 16S rRNA confirmed its position in this genus, with its closest phylogenetic relatives being M. rhodesianum, M. zatmanii and M. extorquens, from which it differed in its ability to grow on several diagnostic substrates. Methanol-grown organisms contained high activities of hydroxypyruvate reductase -3 micromol NADH oxidized min-1 (mg crude extract protein)-1], showing that the serine pathway was used for methylotrophic growth. M. thiocyanatum was able to use thiocyanate or cyanate as the sole source of nitrogen for growth, and thiocyanate as the sole source of sulfur in the absence of other sulfur compounds. It tolerated high concentrations (at least 50 mM) of thiocyanate or cyanate when these were supplied as nitrogen sources. Growing cultures degraded thiocyanate to produce thiosulfate as a major sulfur end product, apparently with the intermediate formation of volatile sulfur compounds (probably hydrogen sulfide and carbonyl sulfide). Enzymatic hydrolysis of thiocyanate by cell-free extracts was not demonstrated. Cyanate was metabolized by means of a cyanase enzyme that was expressed at approximately sevenfold greater activity during growth on thiocyanate [Vmax 634 +/- 24 nmol NH3 formed min-1 (mg protein)-1] than on cyanate [89 +/- 9 nmol NH3 min-1 (mg protein)-1]. Kinetic study of the cyanase in cell-free extracts showed the enzyme (1) to exhibit high affinity for cyanate (Km 0.07 mM), (2) to require bicarbonate for activity, (3) to be subject to substrate inhibition by cyanate and competitive inhibition by thiocyanate (Ki 0.65 mM), (4) to be unaffected by 1 mM ammonium chloride, (5) to be strongly inhibited by selenocyanate, and (6) to be slightly inhibited by 5 mM thiosulfate, but unaffected by 0.25 mM sulfide or 1 mM thiosulfate. Polypeptides that might be a cyanase subunit (mol.wt. 17.9 kDa), a cyanate (and/or thiocyanate) permease (mol.wt. 25.1 and 27.2 kDa), and a putative thiocyanate hydrolase (mol.wt. 39.3 kDa) were identified by SDS-PAGE. Correlation of the growth rate of cultures with thiocyanate concentration (both stimulatory and inhibitory) and the kinetics of cyanase activity might indicate that growth on thiocyanate involved the intermediate formation of cyanate, hence requiring cyanase activity. The very high activity of cyanase observed during growth on thiocyanate could be in compensation for the inhibitory effect of thiocyanate on cyanase. Alternatively, thiocyanate may be a nonsubstrate inducer of cyanase, while thiocyanate degradation itself proceeds by a carbonyl sulfide pathway not involving cyanate. A formal description of the new species (DSM 11490) is given.

The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.