Screening of Bacillus mojavensis biofilms and biosurfactants using laser ablation electrospray ionization mass spectroscopy.

AIMS: Biofilms are composed of micro-organisms within a matrix of chemically complex polymer compounds and from these structures many unknown competitive factors are suggested that many considered are important consequences for biological control. This research was undertaken to study further the endophyte, Bacillus mojavensis and its relationships to biofilm and two classes of lipopeptides considered relevant for biocontrol of plant pathogens. METHODS AND RESULTS: Laser ablation electrospray ionization mass spectrometry and conventional MS/MS were used to study in situ biofilm production and the production of lipopeptides fengycin and surfactin in different strains of B. mojavensis in plate and test tube culture on two media. All strains were capable of producing biofilm in vitro along with the accumulation of surfactin and fengycin although no concentration-dependent relationship between lipopeptide accumulation and biofilm was observed. CONCLUSION: All strains studied produce biofilms in culture with the accumulated surfactin and fengycin, demonstrating that endophytic bacteria also produced biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that this endophytic species produced biofilms along with two biocontrol compounds of which one, surfactin, considered by others as a quorum sensor, highlighting its ecological role as a signalling mechanism in planta.

In situ ergot alkaloid detection in three Balansia epichloe-infected grass species.

AIMS: The objectives of this work were to characterize molecularly the morphologically described endophyte Balansia epichloe symbiotic on three grass species, and to determine the in situ production of ergot alkaloids on these three symbiota. METHODS AND RESULTS: Balansia epichloe symbiotic with smut grass (Sporobolus poiretii), love grass (Eragrostis hirsuta) and lace grass (Eragrostis capillaries, a new host) were characterized using DNA barcoding. Laser ablation electro spray ionization (LAESI)-mass spectrometry was used to detect ergot alkaloids in situ for each symbiotum. CONCLUSIONS: The three morphologically described symbionts on the three host grasses were indicated as belonging to the species B. epichloe, DNA barcoding suggested they were related although a cryptic species was suggested. LAESI-mass spectrometry showed that ergot alkaloids were produced in vivo in two hosts but not the third although this same symbiotum was related to one of the ergot alkaloid producing symbiota as revealed by the DNA-barcoding procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: These results established the accumulation of ergot alkaloids in pot culture by a morpho species although there were variations with each species of grass. Barcoding described divergence among species, but considering its limitation, the suggested existence of cryptic species among this morphospecies requires substantiation by studies that are more rigorous.

Efficient inhibition of Escherichia coli RNA polymerase by the bacteriophage T4 AsiA protein requires that AsiA binds first to free sigma70.

The bacteriophage T4 AsiA protein inhibits transcription from host and phage early promoters and is required, along with the T4 MotA protein, for activation of phage middle promoters. During infection, AsiA is found in a tight association with the sigma70 subunit of RNA polymerase. We show that AsiA binds rapidly to free sigma70 at either 4 degrees C or 30 degrees C to form an AsiA-sigma70 complex that with core efficiently reconstitutes the AsiA-inhibited RNA polymerase. In contrast, AsiA does not inhibit transcription after a 15 minute incubation with RNA polymerase holoenzyme at 4 degrees C, and at 30 degrees C an incubation of several minutes is required to inhibit most of the polymerase. We show that the heat step needed for AsiA is not the formation of an active AsiA protein. However, it is consistent with the momentary dissociation of holoenzyme to give free sigma70 and core. Our results indicate that AsiA is either unable to access holoenzyme directly or does so very slowly. Efficient generation of the AsiA-inhibited RNA polymerase requires that AsiA first binds to free sigma70 and then the AsiA-sigma70 complex binds to core to form the Asi-A-inhibited polymerase.

Specific in vitro transcription of the insertion sequence IS2.

The insertion sequence IS2 is a small transposable element of Escherichia coli that lacks any known genetic markers. Insertion of this element in one orientation (I) within bacterial operons blocks expression of downstream genes. In the other orientation (II), IS2 has been associated with the constitutive expression of genes distal to its insertion, suggesting that IS2 might contain promoters directing transcription of IS2(II) into other genes. To test the transcription potential of IS2, we have transcribed in vitro DNA templates from gal3, a Gal- allele in which an IS2(I) is inserted between the gal promoter and the gal genes. We have detected two IS2-specific RNAs which initiate from promoters within IS2 and are transcribed in orientation II (away from the galETK genes). Though the presence and orientation of these promoters suggests that they could be responsible for the constitutive expression of genes adjacent to an IS2(II) element, an alternative role could be for transcription of IS2-encoded genes. Although IS2(I) insertions normally block expression of adjacent genes, certain altered (e.g. mutant) IS2(I) sequences lead to the constitutive expression of downstream genes. We have transcribed DNA templates from galwc5 and galc331, which are Galc alleles that contain altered IS2(I) insertions within the gal operon. For each allele, we have detected two gal-directed transcripts initiating within the IS2 sequence. These RNAs are not detected upon transcription of the unaltered IS2(I) DNA and the promoters arise as a direct consequence of the IS2(I) alterations. This result suggests that these promoters detected in vitro are responsible for the Galc phenotype of these alleles.

Binding of the bacteriophage T4 transcriptional activator, MotA, to T4 middle promoter DNA: evidence for both major and minor groove contacts.

During infection, the bacteriophage T4 transcriptional activator MotA, the co-activator AsiA, and host RNA polymerase are needed to transcribe from T4 middle promoters. Middle promoters contain a -10 region recognized by the sigma(70)subunit of RNA polymerase and a MotA box centered at -30 that is bound by MotA. We have investigated how the loss or modification of base determinants within the MotA box sequence 5'TTTGCTTTA3' (positions -34 to -26 of a middle promoter) affects MotA function. Gel retardation assays with mutant MotA boxes are consistent with the idea that MotA uses minor groove contacts upstream and major groove contacts downstream of the center GC, and does not require any specific base feature at the C.G base-pair at position -30. In particular, the 5-methyl residue on the thymine residue at position -29, a major groove contact, contributes to MotA binding, while converting the T.A at -32 to a C. I base-pair, a change that affects the major but nor the minor groove, yields a MotA box that is similar to wild-type. However, methylation interference analyses indicate that neither the binding of MotA nor the binding of polymerase/MotA/AsiA to the middle promoter PuvsXis inhibited by premethylation of guanine and adenine residues, suggesting that binding does not require minor groove contact with any specific T.A base-pair. Using gel retardation analyses, we calculate an apparent dissociation constant of 130 nM for MotA binding to the wild-type MotA box. Previous work has shown that the N-terminal region of MotA is needed for an interaction between MotA and sigma(70). We suggest that this MotA-sigma(70)interaction helps to stabilize the relatively weak interaction of MotA with the -30 region of middle promoter DNA.

The bacteriophage T4 transcriptional activator MotA accepts various base-pair changes within its binding sequence.

During infection, bacteriophage T4 regulates three sets of genes: early, middle, and late. The host RNA polymerase is capable of transcribing early genes, but middle transcription requires the T4-encoded transcriptional activator, MotA protein, and the T4 co-activator, AsiA protein, both of which bind to the sigma 70 (sigma70) subunit of RNA polymerase. MotA also binds a DNA sequence (a MotA box), centered at position -30. The identification of more than 20 middle promoters suggested that a strong match to the MotA box consensus sequence (t/a)(t/a)TGCTT(t/c)A was critical for MotA activation. We have investigated how specific base changes within the MotA box sequence affect MotA binding and activation in vitro, and we have identified seven new middle promoters in vivo. We find that an excellent match to the sigma70 -10 consensus sequence, rather than an excellent match to the MotA box consensus sequence, is an invariant feature of MotA-dependent promoters. Many single base changes in the MotA box are tolerated in binding and activation assays, indicating that there is more flexibility in the sequence requirements for MotA than was previously appreciated. We also find that using the natural T4 DNA, which contains glucosylated, 5-hydoxymethylated cytosine residues, affects the ability of particular MotA box sequences to activate transcription. We suggest that MotA and AsiA may function like certain eukaryotic TAFs (TATA binding protein (TBP) associated factors) whose binding to TBP results in transcription from new core promoter sequences.

Characterization of pre-transcription complexes made at a bacteriophage T4 middle promoter: involvement of the T4 MotA activator and the T4 AsiA protein, a sigma 70 binding protein, in the formation of the open complex.

Bacteriophage T4 middle promoters have excellent matches to the -10 consensus sequence for the sigma 70 subunit of Escherichia coli RNA polymerase, but a binding site for the T4 transcriptional activator MotA replaces the sigma 70 -35 consensus. E. Coli RNA polymerase transcribes from middle promoters with or without the activator. In contrast, transcription by T4-modified E. coli RNA polymerase, which is present during T4 infection, requires NotA. We show that transcription by unmodified polymerase from the T4 middle promoter P uvsx is independent of the specific sequences within the -35 region, and the Dnase I footprint obtained with unmodified polymerase and P uvsx resembles those seen previously with E. coli extended -10" promoters. In contrast, although T4-modified polymerase alond binds P uvsx, promoter unwinding and detection of a Dnase I footprint requires MotA. This footprint is significantly different from that obtained with unmodified polymerase, starting upstream of around position -20. Previous work has indicated that the T4 AsiA protein, which binds tightly to sigma 70, is the phage modification required for MotA activation. We show that in the presence of AsiA, MotA, and otherwise unmodified polymerase, Dnase I protection of P uvsx is now similar to that obtained with the fully modified polymerase and MotA up to around position -40. However, protection upstream of -40 is still similar to that seen with unmodified polymerase. Our results support the idea that MotA-dependent activation requires AsiA binding to sigma 70 to achieve specific protein-DNA contacts within the -20 to -40 region of a middle promoter.

Domain 1.1 of the sigma(70) subunit of Escherichia coli RNA polymerase modulates the formation of stable polymerase/promoter complexes.

The sigma 70 (sigma(70)) subunit of Escherichia coli RNA polymerase specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma(70) is present within polymerase holoenzyme, two of its domains, 2.4 and 4.2, interact with sequences within the -10 and -35 regions, respectively, of promoter DNA. However, in free sigma(70), DNA binding is prevented by domain 1.1, the N-terminal domain of the protein. Previous work has demonstrated that the presence of domain 1.1 is required for efficient transcription initiation at the lambda promoter P(R). To investigate whether this is a general property of domain 1.1, we have used five promoters to compare polymerases with and without domain 1.1 in in vitro transcription assays, and in assays assessing the formation and decay of stable, pretranscription complexes. We find that the absence of domain 1.1 does not render the polymerase defective at all of these promoters. Depending on the promoter, the absence of domain 1.1 can promote or inhibit transcription initiation by affecting the formation of stable pretranscription complexes. However, domain 1.1 does not affect the stability of these complexes once they are formed. For polymerases containing domain 1.1, the efficiency of stable complex formation correlates with how well the -10 and -35 regions of a promoter match the ideal sigma(70) recognition sequences. However, when domain 1.1 is absent, having this match becomes less important in determining how efficiently stable complexes are made. We suggest that domain 1.1 influences initiation by constraining polymerase to assess a promoter primarily by the fitness of its -10 and -35 regions to the canonical sequences.

Biological control of Fusarium moniliforme in maize.

Fusarium moniliforme Sheldon, a biological species of the mating populations within the (italic)Gibberella fujikuroi species complex, i.e., population A [= G. moniliformis (Sheld.) Wineland], is an example of a facultative fungal endophyte. During the biotrophic endophytic association with maize, as well as during saprophytic growth, F. moniliforme produces the fumonisins. The fungus is transmitted vertically and horizontally to the next generation of plants via clonal infection of seeds and plant debris. Horizontal infection is the manner by which this fungus is spread contagiously and through which infection occurs from the outside that can be reduced by application of certain fungicides. The endophytic phase is vertically transmitted. This type infection is important because it is not controlled by seed applications of fungicides, and it remains the reservoir from which infection and toxin biosynthesis takes place in each generation of plants. Thus, vertical transmission of this fungus is just as important as horizontal transmission. A biological control system using an endophytic bacterium, Bacillus subtilis, has been developed that shows great promise for reducing mycotoxin accumulation during the endophytic (vertical transmission) growth phase. Because this bacterium occupies the identical ecological niche within the plant, it is considered an ecological homologue to F. moniliforme, and the inhibitory mechanism, regardless of the mode of action, operates on the competitive exclusion principle. In addition to this bacterium, an isolate of a species of the fungus Trichoderma shows promise in the postharvest control of the growth and toxin accumulation from F. moniliforme on corn in storage.

Pericardial-fluid complement: normal values.

Reports of low pericardial-fluid complement levels in systemic lupus erythematosus and rheumatoid arthritis have been difficult to interpret, as few data are available to describe complement concentrations in patients without pericardial disease. The authors therefore determined normal values under standardized conditions of collection, storage, and assay. The normal ranges for pericardial-fluid C3, C4, and total hemolytic complement were 35-127 mg/dl, 6.3-23 mg/dl, and 1.9-9.1 CH50 units, respectively. Storage at -20 C resulted in a 50% reduction in values. Hence, storage at -70 C is recommended. As the level of pericardial-fluid total hemolytic complement is normally low, caution is needed in interpreting its apparent reduction in various immunologic diseases.

Temporal patterns of DNA adduct formation and glutathione S-transferase activity in the testes of rats fed aflatoxin B1: a comparison with patterns in the liver.

Fisher-344 male rats were fed 1.6 ppm of aflatoxin B1 (AFB1) continuously and intermittently for several weeks. At various time periods, DNA was isolated from the testes and livers and analyzed for AFB1-DNA adducts. The ability of the testis to detoxify AFB1 was also investigated by the glutathione S-transferase (GST) activity assay and compared with that of the liver. The levels of testicular AFB1-DNA adducts were 2.4 to 8.1 times lower than those of the liver after 4 to 16 weeks of continuous treatment and 2.2 to 46.2 times lower after 8 to 20 weeks of intermittent treatment. The testicular DNA adducts markedly decreased over time. By 16 weeks of continuous and 20 weeks of intermittent exposure, they had decreased 37 and 91%, respectively. In contrast, hepatic AFB1-DNA adducts increased four-fold from 4 to 16 weeks of continuous treatment but increased at a much slower rate after intermittent exposure. In both the liver and testis, significant levels of AFB1-DNA adducts persisted for at least 1 month after ending the treatment, suggesting that this type of lesion was poorly repaired. In control rats, the testis showed significantly higher GST activity than the liver. In treated rats, these differences were significant during the first 12 weeks of continuous treatment but not at later times. Tissue-specific differences such as germ-cell depletion and increased testicular detoxification may play an important role in the observed differential pattern of DNA adduct formation between the testis and liver.

Effects of cyclopiazonic acid on the ultrastructure of rat liver.

Groups of Sprague-Dawley rats were dosed per os for 4 consecutive days with 0.0, 0.2, 2.0 or 4.0 mg cyclopiazonic acid (CPA)/kg body weight/day, and killed on the fifth day. Sections of liver were prepared for electron microscopic examination. Dilatation of the rough endoplasmic reticulum was observed in all hepatocytes examined from the 2 highest dose groups, and in about 25% of liver cells from the 0.2 mg CPA/kg/day group. Vesiculation of the rough endoplasmic reticulum also occurred in these groups, an increasing amount of vesiculation being observed with increasing dosage. Control sections exhibited neither of these characteristics. No proliferation of smooth endoplasmic reticulum, or blockage of bile canaliculi was observed in any group. Lysing cells were present only in the 4.0 mg CPA/kg/day group; mitochondria in the 2.0 and 4.0 mg CPA/kg/day dose groups were swollen. Nuclei were ultrastructurally normal in all groups. The primary cellular effect of CPA was on the endoplasmic reticulum, even at relatively low doses. Possible interactions of CPA with other toxins likely to be produced by the same fungus, such as aflatoxin, are considered.

Combined effects of the mycotoxins aflatoxin B1 and cyclopiazonic acid on Sprague-Dawley rats.

This study was conducted to determine whether exposure to cyclopiazonic acid (CPA) and aflatoxin B1 (AFB1) would alter the toxicity associated with exposure to either toxin individually. Groups of male rats were administered 0, 0.1 or 4.0 mg CPA/kg body weight/day intragastrically (three groups per dose level) for three consecutive days and 30 min after each of these CPA doses the rats were dosed by gavage with 0, 0.1 or 2.0 mg AFB1/kg body weight/day. Six of the 12 rats given each of these nine treatments were killed on day 4 after the initial dosing, and the rest were allowed a recovery period of 4 days prior to being killed. Weight loss in the three groups receiving 2.0 mg AFB1/kg/day occurred within 24 hr of the first doses. Feed consumption by these rats was about 60% of that in the other groups. By the end of the recovery period, rats in these three groups had lost an average of 31-38 g. Feed consumption throughout the recovery period by rats in the 2.0-mg AFB1 groups was about 50% of the control value, except in the group that also received the high dose of CPA, in which it was 75%. Gross pathological findings were primarily limited to rats in the high AFB1 group, and included icterus, shrunken liver and lesions in the kidney at the cortico-medullary junction. Microscopic changes were characteristic of aflatoxicosis in rats. Glycocholic acid assays indicated liver damage only in those groups that received the high AFB1 dose. We conclude that neither toxin potentiates the action of the other at the dose levels used in this study.

Fusaric acid and pathogenic interactions of corn and non-corn isolates of Fusarium moniliforme, a nonobligate pathogen of corn.

Fusarium moniliform is a nonobligate parasite of corn, which exists as a complex of closely related fungi from different mating population or biological species. Strains of this fungus isolated from corn, have been determined to belong to mating populations A, although other populations have been isolated from corn. The ultrastructural association of the fungus with corn during growth, and the effects of the host on suppression of disease suppression are reviewed. This fungus enters a relationship with corn cultivars that is not always pathogenic. Pathogenesis is delayed, if it ever occurs. F. moniliforme can exist entirely as an endophyte, systemically colonizing kernels, remaining there until germination upon which the fungus infects the emerging seedlings. The symptomless association persists during the growth cycle of corn, and the resulting endophytic hyphae may be the source of mycotoxin production. The host's ability to suppress the fungus appears to be related to one class of compounds, the cyclic hydroxamic acids and their decomposition products, which can be catabolized by the fungi of mating population A but not C.

Effects of Endophytic Infection by Fusarium moniliforme on Corn Growth and Cellular Morphology.

Kernels of corn, Zea mays, were inoculated with Fusarium moniliforme to analyze seedling growth and development during endophytic, symptomless infection. In planta F. moniliforme distribution and seedling growth, expressed as shoot diameter, plant height, leaf length, and dry weight, were examined weekly for 28 days after planting. Even though no visible disease symptoms developed, F. moniliforme was isolated from most segments taken from seedlings grown from inoculated, but not noninoculated, kernels from the earliest to the latest sampling. F. moniliforme did not alter the rate or percentage of kernel germination, but seedlings grown from inoculated kernels had suppressed shoot diameter, plant height, leaf length, and plant weight 7 days after planting. However, seedling growth from inoculated kernels was similar to or greater than that from noninoculated kernels at 28 days. Histological modifications in seedlings grown from inoculated kernels included accelerated lignin deposition in shoots and modified chloroplast orientation in leaves. In summary, gross morphology and histology were altered in corn seedlings during symptomless, endophytic infection by F. moniliforme.