Nutrient-limited Medium Research Articles

Inducer Bacteria, Unique Signal Peptides, and Low-Nutrient Media Stimulate in Vitro Bacteriocin Production by Lactobacillus Spp. and Enterococcus Spp. Strains

Bacteriocins (BCN) are antimicrobial peptides that provide potential to control bacterial infections in a variety of applications. We previously reported on three class IIa BCN molecules produced by Lactobacillus salivarius B-30514 (OR-7), Enterococcus faecium B-30746 (E 50-52), and Enterococcus durans/faecium/hirae B-30745 (E-760). These BCN are notably effective against a wide array of pathogenic bacteria. To commercially apply such BCN, adequate quantities must be produced and harvested. We determined that a combination of host producer synthesized signal peptides (SP) in the presence of both producer and inducer bacteria grown in a dilute fermentation medium enabled marked increases in the synthesis of BCN. These SP contained 24-30 amino acid residues with sizes ranging from 2095 to 3065 Da having the unique terminal carboxyl sequence of VKGLT. The inducer bacterial isolates used were Lactobacillus acidophilus B-30510 and Lactobacillus crispatus B-30884. We used a nutrient-limited medium of 10% brucella broth (containing 0.01% glucose) to enhance BCN production. Using the combination of these above three parameters enabled us to reproducibly harvest at least 200 mg of BCN/L of the spent fermentation broth. This information can be used to assist in the production of BCN.

Novel Cyclic Hexapeptides from Marine-Derived Fungus, Aspergillus sclerotiorum PT06-1

Two novel cyclic hexapeptides containing both anthranilic acid and dehydroamino acid units, sclerotides A (1) and B (2), were isolated from the marine-derived halotolerant Aspergillus sclerotiorum PT06-1 in a nutrient-limited hypersaline medium. Both 1 and 2 are photointerconvertible and could be interconverted via a radical reaction initiated by direct photoisomerization. Both compounds showed moderate antifungal activity. Compound 2 also showed weak cytotoxicity and antibacterial activity.

Role of the Hya Hydrogenase in Recycling of Anaerobically Produced H 2 in Salmonella enterica Serovar Typhimurium

Double and triple uptake-type hydrogenase mutants were used to determine which hydrogenase recycles fermentatively produced hydrogen. The Deltahyb Deltahya and Deltahyd Deltahya double mutants evolved H(2) at rates similar to that of the triple mutant strain, so Hya alone oxidizes the bulk of H(2) produced during fermentation. When only Hya was present, no hydrogen production was observed in nutrient-limited medium. H(2) uptake assays showed that Hya can oxidize both exogenously added H(2) and formate hydrogen lyase-evolved H(2) anaerobically. Even after anaerobic growth, all three uptake-type hydrogenases could function in the presence of oxygen, including using O(2) as a terminal acceptor.

Effect of Different Incubation Atmospheres on the Production of Biofilm in Methicillin-Resistant Staphylococcus aureus (MRSA) Grown in Nutrient-Limited Medium

Today, methicillin-resistant Staphylococcus aureus (MRSA) is a feared cause of nosocomial infections worldwide. These organisms can gain increased resistance to antimicrobial agents through biofilm formation, which appears to be a bacterial survival strategy. MRSA isolates obtained from patients were cultured in nutrient-limited medium supplemented with 0.2% glucose in aerobic, anaerobic, and CO(2) incubation atmospheres. Biofilm formation was quantified by the microtiter plate test. MRSA strains showed significantly lower biofilm production when grown in an aerobic atmosphere compared to that exhibited in CO(2)-rich environments. Gaseous conditions and growth in a nutritionally limited medium can profoundly influence the amount of biofilm formation in MRSA. This should be considered in any in vitro study of in vivo behavior.

Presence and potential for horizontal transfer of antibiotic resistance in oxidase-positive bacteria populating raw salad vegetables

To assess whether domestically grown fresh salad vegetables constitute a possible reservoir of antibiotic resistance for Canadian consumers, aerobic bacteria capable of forming colonies at 30 °C on nutrient-limited media were recovered from a single sampling of Romaine lettuce, Savoy spinach and alfalfa sprouts, then examined for their susceptibility to ten antibiotics and the carriage of potentially mobile R-plasmids and integrons. Of the 140 isolates resistant to one or more antibiotic, 93.5 and 90.0% were resistant to ampicillin and cephalothin; 35.7% to chloramphenicol, 10.0% to streptomycin, 4.2% to nalidixic acid, 4.2% to kanamycin, and 2.8% to gentamicin. Gram-positive isolates accounted for less than 4% of the antibiotic resistant strains. A small portion (23.1%) of the predominant oxidase-positive, gram-negative isolates was resistant to two or more antimicrobials. Members of the Pseudomonas fluorescens/putida complex were most prevalent among the 34 resistant strains identified. Sphingobacterium spp. and Acinetobacter baumanni also were detected. Ten of 52 resistant strains carried plasmids, 3 of which were self-transmissible and bore resistance to ampicillin and kanamycin. Eighteen of 48 gave PCR evidence for integron DNA. Class 2 type integrons were the most prevalent, followed by class 1. We conclude that the foods examined here carry antibiotic resistant bacteria at the retail level. Further, our determination that resistant strains contain integron-specific DNA sequences and self-transmissible R-plasmids indicates their potential to influence the pool of antibiotic resistance in humans via lateral gene transfer subsequent to ingestion.

Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate- co -3-Hydroxyvalerate) Synthase in Haloferax mediterranei

The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC(Hme)) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE(Hme) and phaC(Hme) genes were constitutively expressed, and both the PhaE(Hme) and PhaC(Hme) proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC(Hme) genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC(Hme) genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaC(Hme) was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaE(Hme)/PhaC(Hme) (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaC(Hme) and PhaE(Hme), accounted for the PHBV synthesis in H. mediterranei.

The tyrosine degradation gene hppD is transcriptionally activated by HpdA and repressed by HpdR in Streptomyces coelicolor, while hpdA is negatively autoregulated and repressed by HpdR

Streptomyces coelicolor produces a brown pigment on nutrient-limited agar medium (Tyr-PM) using l-tyrosine as the sole nitrogen and carbon source. The pigment production is associated with the second step of l-tyrosine catabolism catalysed by 4-hydroxyphenylpyruvate dioxygenase (HppD), which converts 4-hydroxyphenylpyruvate (4HPP) to 2, 5-dihydroxyphenylacetate (homogentisate) to provide the carbon and energy substrates for the growth of S. coelicolor on Tyr-PM. An hppD mutant did not produce brown pigment, and its normal growth was impaired on Tyr-PM. hpdA and hpdR, located close to hppD, were identified as activator and repressor genes for hppD transcription in the presence of tyrosine. hpdA, divergently transcribed from hppD, is negatively autoregulated in the absence of tyrosine, whereas it is repressed by both its own protein and HpdR in the presence of tyrosine. Electrophoretic mobility shift assays and footprinting experiments showed that HpdA and HpdR each bind to an overlapping region spanning the promoters of both hppD and hpdA, and that 4HPP, instead of tyrosine, is the specific ligand modulating the binding patterns and footprints of HpdA and HpdR on the hppD-hpdA promoter region. These results suggested that the transcription of hppD is subject to coarse and fine control by a complex regulatory system.

Expressed Sequence Tags from Phytophthora sojae Reveal Genes Specific to Development and Infection

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.

Effects of Various Growth Conditions in a Chemostat on Expression of Virulence Factors inPorphyromonas gingivalis

Porphyromonas gingivalis, one of the gram-negative organisms associated with periodontal disease, possesses potential virulence factors, including fimbriae, proteases, and major outer membrane proteins (OMPs). In this study, P. gingivalis ATCC 33277 was cultured in a chemostat under hemin excess and presumably peptide-limiting conditions to better understand the mechanisms of expression of the virulence factors upon environmental changes. At higher growth rates, the amounts of FimA and the 75-kDa protein, forming long and short fimbriae, respectively, increased significantly, whereas gingipains decreased in amount and activity. In a nutrient-limited medium, lesser amounts of the above two fimbrial proteins were observed, whereas clear differences were not found in the amounts of gingipains. In addition, two-dimensional electrophoresis revealed that proteins in cells were generally fewer in number during nutrient-limited growth. Under aeration, a considerable reduction in gingipain activity was found, whereas several proteins associated with intact cells significantly increased. However, the expression of major OMPs, such as RagA, RagB, and the OmpA-like proteins, was almost constant under all conditions tested. These results suggest that P. gingivalis may actively control expression of several virulence factors to survive in the widely fluctuating oral environment.

Biofilm-Spore Response in Bacillus cereus and Bacillus subtilis during Nutrient Limitation

This study aimed to trace the dynamics of biofilm formation by vegetative cells and endospores of Bacillus cereus DL5 and Bacillus subtilis 168. Counts of B. cereus DL5 and B. subtilis 168 vegetative cells and spores either attached to glass wool or, correspondingly, planktonic cells were determined by standard plate-counting methods. Results from this study highlighted the biofilm-forming potential of both spores and vegetative cells of two different Bacillus species. It was shown that once Bacillus spores had attached to a surface, the spores germinated under favorable (B. cereus DL5) and even unfavorable (B. subtilis 168) nutrient conditions, resulting in biofilms containing both spores and vegetative populations. Furthermore, it was suggested that vegetative B. cereus DL5 cells exhibited a low propensity for spore formation in attached and planktonic growth forms in nutrient-limited growth medium. By contrast, vegetative B. subtilis 168 cells readily formed spores in planktonic and attached microcosms when exposed to nutrient-limited growth conditions. Sporulation in attached Bacillus populations is an important practical consideration for many food industries, such as dairy processing, where bacilli are routinely isolated from populations attached to processing-equipment surfaces.

Analyses of the Red-Dry-Rough Phenotype of an Escherichia coli O157:H7 Strain and Its Role in Biofilm Formation and Resistance to Antibacterial Agents

In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of Salmonella. In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of Salmonella.

A Combination of Direct Viable Counting, Fluorescence in situ Hybridization, and Green Fluorescent Protein Gene Expression for Estimating Plasmid Transfer at the Single Cell Level

Plasmid-mediated conjugation is an important mechanism through which bacteria establish new genetic traits in the environment. Here we used a combination of direct viable counting (DVC), fluorescence in situ hybridization (FISH), and green fluorescent protein (GFP) gene expression to estimate plasmid transfer frequencies (TFs) on nutrient-limited media at the single cell level. Conjugation experiments on nutrient-rich and -limited media were carried out using Pseudomonas fluorescens as a donor of a broad-host-range plasmid (RK2) tagged with the GFP gene and P. putida as the recipient. FISH-GFP and DVC-FISH-GFP allowed for the accurate detection of donor, recipient and transconjugants by fluorescence microscopy. The TFs obtained by these culture-independent approaches were 1 to more than 3 orders of magnitude higher than those determined by selective plate-counting. Lower and variable TFs obtained by selective plate-counting emphasize the importance of culture-independent approaches in plasmid transfer studies. The application of DVC before FISH-GFP on nutrient-limited medium produced elongated and/or fattened cells and resulted in a better fluorescence signal in target bacteria cells and better accumulation, maturation and fluorescence-emission of GFP within transconjugant cells, facilitating the detection and identification of cells compared with FISH-GFP alone. The DVC-FISH-GFP could be useful for other conjugation studies using bacteria with low metabolic activity under oligotrophic/stressful conditions or studies which require a precise determination of plasmid transfer events during short-term conjugation experiments.

Extracellular Polymer Produced in the Presence of Copper Minerals

A qualitative and semiquantitative assay was applied to estimate production of peripheral exopolymers by strains of bacteria isolated from flotation wastes during growth on copper sulphides and copper minerals wastes at neutral pH. Exopolymer production, expressed as percentage of Alcian Blue adsorption per μ g of cell protein, was the lowest on a complete organic medium and the highest on a nutrient-limited mineral medium. The exopolymer was highly resistant to acid hydrolysis and contained hydrocarbons, saturated and unsaturated fatty acids, aromatic acids and aromatic acid esters, which are well known as artificial surfactants and plasticizers. Carbohydrates appeared incidentally and in trace amount only. Chemical components of the exopolymer or chemically similar compounds also were found in black shale particles in the flotation wastes.

The quormone degradation system of Agrobacterium tumefaciens is regulated by starvation signal and stress alarmone (p)ppGpp.

A unique signal degradation system has recently been discovered in Agrobacterium tumefaciens. Upon entering stationary phase, A. tumefaciens terminates quorum sensing-dependent Ti-plasmid conjugation by degradation of acyl homoserine lactone (AHL) quormone via the enzyme AttM (AHL-lactonase). attM, together with attK and attL, constitute one transcriptional unit subjected to the control of a common promoter. AttJ, the other member of the signal degradation system, is an IclR-like negative transcriptional factor, which tightly represses the expression of AttM at the early stage of bacterial growth. In this study, we found that this quormone degradation system is activated by either carbon or nitrogen starvation. Quormone degradation was significantly delayed when bacterial culture was supplemented with extra carbon or nitrogen source in the nutrient-limited minimal medium before the onset of stationary phase. To identify the signalling pathway and regulatory mechanisms that mediate quormone degradation, we constructed a reporter strain A6(attKLM::lacZ) in which the promoterless lacZ was transcriptionally fused to the attKLM promoter. Transposon mutagenesis of strain A6(attKLM::lacZ) led to identification of the relA gene, which encodes the stress alarmone (p)ppGpp synthetase. Tn5 knock-out of relA abolished the stationary phase-dependent expression of attM. We concluded that the A. tumefaciens quormone degradation system is coupled to and regulated by the generic (p)ppGpp stress response machinery.

Cellular differentiation in response to nutrient availability: The repressor of meiosis, Rme1p, positively regulates invasive growth in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the transition from a nutrient-rich to a nutrient-limited growth medium typically leads to the implementation of a cellular adaptation program that results in invasive growth and/or the formation of pseudohyphae. Complete depletion of essential nutrients, on the other hand, leads either to entry into a nonbudding, metabolically quiescent state referred to as G0 in haploid strains or to meiosis and sporulation in diploids. Entry into meiosis is repressed by the transcriptional regulator Rme1p, a zinc-finger-containing DNA-binding protein. In this article, we show that Rme1p positively regulates invasive growth and starch metabolism in both haploid and diploid strains by directly modifying the transcription of the FLO11 (also known as MUC1) and STA2 genes, which encode a cell wall-associated protein essential for invasive growth and a starch-degrading glucoamylase, respectively. Genetic evidence suggests that Rme1p functions independently of identified signaling modules that regulate invasive growth and of other transcription factors that regulate FLO11 and that the activation of FLO11 is dependent on the presence of a promoter sequence that shows significant homology to identified Rme1p response elements (RREs). The data suggest that Rme1p functions as a central switch between different cellular differentiation pathways.

Nutrient limitation on a planktonic rotifer: Life history consequences and starvation resistance

Nutrient content of plants is low relative to that of herbivores. Dietary nutrients can limit the growth and reproduction of herbivores. We studied life history consequences and starvation resistance for a common planktonic rotifer, Keratella cochlearis, feeding on algae grown on nutrient‐limited media. A strain of Cryptomonas was grown on three types of growth medium with nitrogen (N) and phosphorus (P) adjusted to produce N‐limiting, P‐limiting, and nutrient‐sufficient conditions. The analysis of nutrient content of Cryptomonas cells grown on nutrient‐limited conditions showed that those algae exhibited higher P content than algae growing on a nutrient‐sufficient medium. However, Cryptomonas reached lower densities in nutrient‐limited media. The life history responses of Keratella to food quality were examined with low and high food availability. Our study showed that the interaction of food quantity and quality had a significant effect on rotifer growth rates. We observed, at low food levels, that Keratella growth rates were highest when the algae were grown under P limitation. The lowest rotifer growth rates were obtained when the rotifer fed on Cryptomonas grown in nutrient‐sufficient conditions. Our study also showed that starvation resistance was higher for those rotifers fed on N‐limited Cryptomonas. In natural situations, rotifers could even grow better under nutrient‐limited conditions if this group of algae is dominant in the phytoplankton community. Moreover, differences in starvation resistance could be critical in determining competitive outcome and community structure in nutrient‐variable environments.

Biodegradation of methyl tertiary butyl ether (MTBE) by a bacterial enrichment consortia and its monoculture isolates

Methyl tertiary butyl ether (MTBE), an important gasoline additive, is a recalcitrant compound posing serious environmental health problems. In this study, MTBE-degrading bacteria were enriched from five environmental samples. Enrichments from Stewart Lake sediments and an MTBE contaminated soil displayed the highest rate of MTBE removal; 29.6 and 27.8% respectively, in 28 days. A total of 12 bacterial monocultures isolated from enrichment cultures were screened for MTBE degradation in liquid cultures. In a nutrient-limited medium containing MTBE as the sole source of carbon and energy, the highest rate of MTBE elimination was achieved with IsoSL1, which degraded 30.6 and 50.2% in 14 and 28 days, respectively. In a nutrient-rich medium containing ethanol and yeast extract, the bacterium (Iso2A) substantially removed MTBE (20.3 and 28.1% removal in 14 and 28 days, respectively). Based upon analysis of the 16s rRNA gene sequence and data base comparison, IsoSL1 and Iso2A were identified as a Streptomyces sp. and Sphingomonas sp., respectively. The Streptomyces sp. is a new genera of bacteria degrading MTBE and could be useful for MTBE bioremediation.

Apparent toxicity resulting from the sequestering of nutrient trace metals during standard Selenastrum capricornutum toxicity tests

The water accommodated fractions (WAFs) of aqueous mixtures of three lubricant additives showed significant apparent toxicity to the freshwater alga, Selenastrum capricornutum, and experiments were conducted to investigate the hypothesis that toxicity resulted from the removal of one or more essential nutrients from the test medium by the lubricant additives. Algal growth effects were noted at ashless dispersant A concentrations as low as 0.5 mg l −1 and growth was completely inhibited at 100 mg l −1. Algal cells transferred from the 100 mg l −1 WAF of ashless dispersant A to fresh medium at the end of a standard 96-h toxicity test resumed growing at a rate similar to growth in undosed algal medium, indicating that the effect was algistatic rather than algicidal. Fortifying the iron (Fe) and disodium ethylenediaminetetraacetic acid (EDTA) concentrations of the WAF with 200% of the concentrations used in the formulation of algal medium after 24 h of exposure caused a resumption of algal growth at a rate comparable to the control growth, and a resulting EL50 value above 100 mg l −1. Similar effects were observed when the two other lubricant additives were tested at WAF concentrations that completely inhibited algal growth during standard toxicity tests: fortification of a 50 mg l −1 WAF of ZnDTP with 1000% of the Fe and EDTA used in the formulation of algal medium caused a resumption of growth at a rate statistically identical to the control growth, and the fortification of a 2800 mg l −1 WAF of ashless dispersant B with 700% of the entire complement of nutrients used in the formulation of algal medium caused a resumption of growth at a rate comparable to the control. The indirect toxic effect of these lubricant additives to algae results from the sequestration of one or more nutrient metals essential for algal growth. Standard algal toxicity tests with these lubricant additives may, therefore, have little environmental relevance because the complex chemistry of natural waters differs greatly from the nutrient limited algal medium, and the sequestration effect observed in these static tests could be absent under real world conditions.

Infection of Gymnodinium sanguineum by the dinoflagellate Amoebophrya sp.: effect of nutrient environment on parasite generation time, reproduction, and infectivity.

Preliminary attempts to culture Amoebophrya sp., a parasite of Gymnodinium sanguineum from Chesapeake Bay, indicated that success may be influenced by water quality. To explore that possibility, we determined development time, reproductive output, and infectivity of progeny (i.e. dinospores) for Amoebophyra sp. maintained on G. sanguineum grown in four different culture media. The duration of the parasite's intracellular growth phase showed no significant difference among treatments; however, the time required for completion of multiple parasite generations did, with elapsed time to the middle of the third generation being shorter in nutrient-replete media. Parasites of hosts grown in nutrient-replete medium also produced three to four times more dinospores than those infecting hosts under low-nutrient conditions, with mean values of 380 and 130 dinospores/host, respectively. Dinospore production relative to host biovolume also differed, with peak values of 7.4 per 1,000 microm3 host for nutrient-replete medium and 4.8 per 1,000 microm3 host for nutrient-limited medium. Furthermore, dinospores produced by "high-nutrient" parasites had a higher success rate than those formed by "low-nutrient" parasites. Results suggest that Amoebophrya sp. is well adapted to exploit G. sanguineum populations in nutrient-enriched environments.

Polar glycerolipids of Chlamydomonas moewusii

The fatty acid and polar lipid compositions of the unicellular green alga Chlamydomonas moewusii were characterized. Since this organism is an important plant model for phospholipid-based signal transduction, interest was focused on the lipids phosphatidic acid, phosphatidylinositolphosphate and phosphatidylinositolbisphosphate. A phosphatidylinositol:phosphatidylinositolphosphate:phosphatidylinositolbisphosphate ratio of 100:1.7:1.3 was found. The polyphosphoinositides accounted for 0.8 mol% of the total phospholipids and their fatty acid compositions were similar to that of phosphatidylinositol except for the enrichment of linolenic acid in phosphatidylinositol phosphate. Phosphatidic acid accounted for 0.67 mol% of the phospholipids. Major structural glycerolipids were monogalactosyldiacylglycerol (35 mol%), digalactosyldiacylglycerol (15 mol%), sulfoquinovosyldiacylglycerol (10 mol%), diacylglyceryltrimethylhomoserine (16 mol%), phosphatidylglycerol (9 mol%), phosphatidylethanolamine (8 mol%) and phosphatidylinositol (6 mol%). Relative changes in the total fatty acid compositions found during growth on nutrient-limited medium reflected mainly alterations in the compositions of the chloroplast lipids phosphatidylglycerol and monogalactosyldiacylglycerol. [ 32 P ]Pi-incorporation studies revealed that it took 6 days before the amount of label in the major phospholipids was proportional to their abundance.