Control of human telomere length by TRF1 and TRF2.

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.

Seroprevalence of HTLV-I/II and HIV-1 infection among male intravenous drug abusers in Chicago.

We surveyed for serologic evidence of either HIV-1 or HTLV-I/II infection in 387 male veterans who entered into an inpatient drug treatment center. Serum was obtained after receiving written informed consent. Serum specimens were tested by enzyme-linked immunosorbent assay for antibody to HIV-1 and for antibody to HTLV-I/II; sera that were repeatedly reactive were then tested by Western blot (HIV-1/HTLV-I/II) and radioimmunoprecipitation assay (HTLV-I/II). Sixty-five of 387 (16.79%) patients were tested and confirmed as positive for HTLV-I/II only antibodies and 30 of the 387 (7.75%) were positive for HIV-1 only antibodies. An additional nine patients (2.32%) were seropositive for antibodies to both viruses. A statistically significant difference in the CD4/CD8 lymphocyte ratio was associated with HIV-1 seropositivity. HTLV-I/II seropositivity was strongly associated with black race, age, and duration of i.v. drug use, but not with sexual intercourse as determined by lifetime history of number of sexual partners, incidence of sexually transmitted diseases, type of drug used, or needle-sharing practices.

Prevalence of childhood physical abuse in adult male veteran alcoholics.

Although past research has identified psychological and behavioral consequences for adults who were abused as children, few studies have examined the incidence and consequences of childhood physical abuse among adult alcoholics. The purpose of this study was to assess the prevalence of a childhood history of physical abuse in adult male alcoholics and to determine what differences may exist in the psychological profile and patterns of alcohol abuse in abused and nonabused alcoholics. The study sample was comprised of 100 male alcoholic inpatients from the alcoholism treatment unit at a metropolitan Veterans Administration hospital. Subjects were administered a self-report devised by the authors to assess a history of childhood physical abuse, the Michigan Alcoholism Screening Test (MAST), the Severity of Alcohol Dependence Questionnaire (SADQ), and the Symptom Checklist-90-Revised (SCL-90-R). Findings indicated that approximately one-third of the alcoholics were physically abused as children. Abused alcoholics reported having more severe psychological symptoms and distress than their nonabused counterparts, although they did not differ on the onset, severity, or treatment history for alcohol dependency.

Continuous engineering of nano-cocrystals for medical and energetic applications.

Cocrystals, solid mixtures of different molecules on molecular scale, are supposed to be tailor made materials with improved employability compared to their pristine individual components in domains such as medicine and explosives. In medicine, cocrystals are obtained by crystallization of active pharmaceutical ingredients with precisely chosen coformers to design medicaments that demonstrate enhanced stability, high solubility, and therefore high bioavailability and optimized drug up-take. Nanoscaling may further advance these characteristica compared to their micronsized counterparts - because of a larger surface to volume ratio of nanoparticles. In the field of energetic materials, cocrystals offer the opportunity to design smart explosives, combining high reactivity with significantly reduced sensitivity, nowadays essential for a safe manipulation and handling. Furthermore, cocrystals are used in ferroelectrics, non-linear material response and electronic organics. However, state of the art batch processes produce low volume of cocrystals of variable quality and only have produced micronsized cocrystals so far, no nano-cocrystals. Here we demonstrate the continuous preparation of pharmaceutical and energetic micro- and nano-cocrystals using the Spray Flash Evaporation process. Our laboratory scale pilot plant continuously prepared up to 8 grams per hour of Caffeine/Oxalic acid 2:1, Caffeine/Glutaric acid 1:1, TNT/CL-20 1:1 and HMX/Cl-20 1:2 nano- and submicronsized cocrystals.

Characterization of transposon Tn5469 from the cyanobacterium Fremyella diplosiphon.

A transposon, designated Tn5469, was isolated from mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon following its insertion into the rcaC gene. Tn5469 is a 4,904-bp noncomposite transposon with 25-bp near-perfect terminal inverted repeats and has three tandemly arranged, slightly overlapping potential open reading frames (ORFs) encoding proteins of 104.6 kDa (909 residues), 42.5 kDa (375 residues), and 31.9 kDa (272 residues). Insertion of Tn5469 into the rcaC gene in strain FdR1 generated a duplicate 5-bp target sequence. On the basis of amino acid sequence identifies, the largest ORF, designated tnpA, is predicted to encode a composite transposase protein. A 230-residue domain near the amino terminus of the TnpA protein has 15.4% amino acid sequence identity with a corresponding domain for the putative transposase encoded by Lactococcus lactis insertion sequence S1 (ISS1). In addition, the sequence for the carboxyl-terminal 600 residues of the TnpA protein is 20.0% identical to that for the TniA transposase encoded by Tn5090 on Klebsiella aerogenes plasmid R751. The TnpA and TniA proteins contain the D,D(35)E motif characteristic of a recently defined superfamily consisting of bacterial transposases and integrase proteins of eukaryotic retroelements and retrotransposons. The two remaining ORFs on Tn5469 encode proteins of unknown function. Southern blot analysis showed that wild-type F. diplosiphon harbors five genomic copies of Tn5469. In comparison, mutant strain FdR1 harbors an extra genomic copy of Tn5469 which was localized to the inactivated rcaC gene. Among five morphologically distinct cyanobacterial strains examined, none was found to contain genomic sequences homologous to Tn5469.

Cyanobacterial transposons Tn5469 and Tn5541 represent a novel noncomposite transposon family.

A noncomposite transposon, designated Tn5541, was isolated from strain Fd33 of the filamentous cyanobacterium Fremyella diplosiphon UTEX 481. Sequence analysis showed that Tn5541 is structurally and genetically very similar to Tn5469, which is also endogenous to F. diplosiphon. Both Tn5469 and Tn5541 encode homologous forms of an unusual composite transposase and a protein of unknown function. DNA hybridization analysis showed that like Tn5469, Tn5541 was not widely distributed among cyanobacterial genera. A similar analysis showed that Tn5469 and Tn5541 were equally limited to and present as multiple genomic copies in three of six distinct strains comprising the Tolypothrix 1 cluster of heterocyst-forming filamentous cyanobacteria. These and other distinguishing features suggest that Tn5469 and Tn5541 represent a novel noncomposite transposon family.

rpbA controls transcription of the constitutive phycocyanin gene set in Fremyella diplosiphon.

Three gene sets encode alpha and beta subunits of the phycobiliprotein phycocyanin (PC) in the filamentous cyanobacterium Fremyella diplosiphon. The cpcB1A1 set (encodes PC1) is constitutively expressed, whereas the cpcB2A2 set (encodes PC2) is expressed only in red light and the cpcB3A3 set (encodes PC3) is expressed only during sulfur-limited growth. Primary pigment mutant strain FdBM1 is characterized by elevated levels of PC. DNA hybridization analysis showed that like many pigment mutants in our strain collection, strain FdBM1 harbors an extra genomic copy of endogenous transposon Tn5469. By direct cloning from FdBM1 genomic DNA, the extra copy of Tn5469 was localized to an open reading frame, which we have designated the rpbA gene. Complementation experiments correlated rpbA activity to the phenotype of strain FdBM1. The predicted RpbA protein contains two regions resembling the characterized helix-turn-helix motif which is involved in DNA recognition by many bacterial and phage transcription regulator proteins. RNA hybridization analysis showed that relative to the parental strain Fd33, the level of transcripts from cpcB1A1, but not cpcB2A2 or cpcB3A3, was significantly elevated in strain FdBM1. Introduction of the intact rpbA gene into strain FdBM1 restored the cpcB1A1 transcript level to that of strain Fd33. These results suggest that the rpbA gene product functions in controlling constitutive transcription from the cpcB1A1 gene set, possibly as a DNA-binding transcriptional repressor element.

Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids.

The psbA multigene family in Synechococcus sp. strain PCC 7942 encodes two forms of the D1 protein; Form I, the product of psbAI, differs from Form II, the product of psbAII and psbAIII, at 25 of 360 amino acid positions. D1 is essential for photosynthesis as a protein component of the photosystem II reaction center. Antisera were raised against purified hybrid proteins encoded by psbAI-lacZ and psbAIII-lacZ translational gene fusions that contain the unique amino termini of Form I and Form II, respectively. Form specificity of each antiserum was verified by Western analysis using thylakoid membranes from mutant strains containing only Form I or Form II. Western analysis of thylakoid membranes from wild-type cells cultured at different light intensities detected both forms of D1 in the membrane and showed changes in the ratio of the two forms. The D1 composition of the membrane matched predicted ratios of the forms based on differential gene expression: psbAI is expressed highest at low light, and both psbAII and psbAIII are expressed highest at high light. Along a gradient of light intensity from 5 microE. m-2.s-1 to 482 microE.m-2.s-1, the relative amount of Form I in thylakoid membranes decreased 58%, while the relative amount of Form II increased 60%. Maximum detection of Form I coupled with minimum detection of Form II in membranes from cells harvested at light intensities below 390 microE.m-2.s-1 suggests a central role for Form I in photosystem II. Increased incorporation of Form II into the thylakoid membrane occurred at light intensities reported by others to be photoinhibitory, suggesting that Form II serves a role in adaptation to high light.

Differential expression of members of a cyanobacterial psbA gene family in response to light.

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes encoding two forms of the D1 protein: form I, the product of psbAI, differs from form II, the product of both psbAII and psbAIII, at 25 of 360 residues. D1 is essential for photosynthesis as a core component of the photosystem II reaction center. Translational gene fusions between each of the Synechococcus psbA genes and the Escherichia coli lacZ gene were inserted into the chromosome of wild-type Synechococcus sp. at the respective psbA loci to serve as in vivo reporters of psbA expression. beta-Galactosidase activities indicated differential expression of the psbA-lacZ gene fusions related to light availability. Expression of psbAI was 500-fold greater than expression of psbAII and 50-fold greater than psbAIII under similar conditions. As light intensity decreased from 600 microE.m-2.s-1 to 2 microE.m-2.s-1, expression of the psbAI reporter increased eightfold while expression of the psbAII and psbAIII reporters decreased 10-fold, suggesting differential production of the two forms of D1 in photosystem II in response to light availability. Relative levels of psbA-lacZ fusion transcripts directly reflected beta-galactosidase activities in the transformants, although the fusion transcripts were less stable than native psbA messages.

Blue and red light reversibly control psbA expression in the cyanobacterium Synechococcus sp. strain PCC 7942.

The three psbA genes encoding the photosystem II D1 protein in Synechococcus sp. strain PCC 7942 respond differentially to an increase in intensity of white light through transcriptional induction of psbAII and psbAIII and accelerated degradation of psbAI and psbAIII messages. We report that the genes exhibit a novel photoreversible response involving blue and red light that is almost indistinguishable from the high-white light response. Transfer of cells from white to low-fluence blue light caused a decrease in the level of the psbAI message and increased levels of psbAII and psbAIII messages, whereas transfer to red or far-red light had little effect. Five min of blue light was sufficient to trigger psbAII and psbAIII induction; five min of subsequent red irradiation attenuated this response, whereas subsequent green or far-red light (or darkness) had no effect. Response to both high and blue light was insensitive to inhibitors of photosynthetic electron transport. We propose that Synechococcus modulates photosystem II biosynthesis in a variable light environment through a photoreception signal pathway, which is independent of photosystem II activity and which is distinct from red/green-reversible control of chromatically adapting cyanobacteria and the red/far-red-reversible phytochrome of plants.

Double-blind comparison of bromocriptine and placebo in cocaine withdrawal.

Twenty-nine cocaine-dependent male veterans without other drug dependence completed a double-blind controlled, randomly-assigned study examining the efficacy of bromocriptine versus placebo in the management of cocaine abstinence symptomatology. Serum prolactin (PL) and growth hormone (GH) levels were obtained prior to and after the study was completed. Patients were seen daily and completed several self-report questionnaires, including the Symptom Checklist-90-Revised, the Beck Depression Inventory, and a Cocaine Craving Report. The patients were also asked to rate a variety of cocaine withdrawal symptoms. Overall, there did not appear to be any advantage to receiving bromocriptine versus placebo during the first 3 weeks following cocaine use cessation with the possible exception of changes in activity and appetite level. The placebo group showed a statistically significant increase in activity level during the first week in treatment and a significant increase in appetite throughout the study. Patients in both groups showed significant improvement in the other areas assessed, with improvement appearing to progress according to length of treatment. Hyperprolactinemia or abnormal GH levels were not found in this patient sample as a group. Thirty-four of the original 63 patients dropped out of the study. Seventeen received bromocriptine, and 17 received placebo. There was no significant difference between drug groups in incidence of retaining patients in treatment. The high dropout rate may reflect the difficulty incurred in retaining cocaine-dependent patients in treatment.

Severity of alcohol dependence and its relationship to additional psychiatric symptoms in male alcoholic inpatients.

Many alcoholics suffer from distress associated with psychiatric symptoms in addition to alcoholism. Although there may be many such symptoms reported by the alcoholic, there may not be enough in any one category to meet DSM III criteria for an additional psychiatric diagnosis, and consequently these symptoms may not be considered in treatment planning. In addition, the prevalence and severity of psychiatric symptoms may be dependent on the progression of alcoholism. The purpose of this study was to examine whether a relationship exists between patterns of psychiatric symptomatology and the level of severity of alcohol dependence in an inpatient male alcoholic sample, and to describe how this information can be utilized to differentiate large groups of alcoholics into clinical meaningful categories. The study sample was comprised of 100 male alcoholic inpatients from the Alcoholism Treatment Unit at a metropolitan Veterans Administration Hospital. Subjects were administered The Michigan Alcoholism Screening Test (MAST), the Severity of Alcohol Dependence Questionnaire (SADQ), and the Symptom Checklist-90-Revised (SCL-90-R). Correlational analysis of the data supported the hypothesis that a positive relationship exists between the severity of alcohol dependence and the prevalence of additional psychiatric symptom patterns. Partial correlational analysis indicated that the length of the alcoholic's illness and the number of inpatient treatments for alcoholism enhanced the relationship between the severity of alcoholism and most of the symptom scales. The utility of assessing concurrent psychiatric symptomatology as it relates to the severity of alcohol dependence is discussed in relation to treatment planning and prognostic assessment of the patient.

DNA-Binding properties of the Fremyella diplosiphon RpbA repressor.

Mutant strain FdBM1 of the cyanobacterium Fremyella diplosiphon is characterized by elevated transcription of the cpcB1A1 gene set due to inactivation of rpbA by Tn5469. The predicted RpbA protein contains two regions resembling the characterized helix-turn-helix (HTH) motif involved in DNA recognition by many phage and bacterial transcription regulator proteins. It was therefore hypothesized that RpbA functions as a DNA-binding repressor involved in the control of transcription from cpcB1A1. A histidine-tagged form of RpbA, designated RpbA-His(6), was examined for its ability to bind to the defined promoter region for cpcB1A1. Gel mobility shift assays showed that RpbA-His(6) specifically binds to a DNA fragment containing the cpcB1A1 promoter and that significant binding can be achieved with equimolar amounts of RpbA-His(6) and the cpcB1A1 promoter probe. DNase I footprint analysis localized the RpbA-His(6) binding site to an asymmetric 21-bp region that overlaps the putative -10 promoter sequence. A mutational analysis suggested that binding by RpbA-His(6) to its cognate DNA may involve both putative HTH motif-like regions. We conclude that RpbA functions as a transcriptional repressor for cpcB1A1 and suggest that binding by RpbA to its cognate DNA may represent an atypical protein-DNA interaction.

Different and rapid responses of four cyanobacterial psbA transcripts to changes in light intensity.

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes which encode two forms of the D1 protein of photosystem II. Experiments using psbA-lacZ translational fusions and Western blot (immunoblot) analysis have shown that the psbA genes respond differently to changes in light intensity, altering the ratio of the two forms of D1 in the thylakoid membrane. Each gene produces a 1.2-kilobase (kb) mRNA. A probe specific for psbAII transcripts also identified a 1.6-kb mRNA which starts 419 base pairs upstream of the 5' end of the 1.2-kb species and overlaps the entire 1.2-kb transcript. This 419-base-pair region includes an open reading frame (ORF1) of 114 amino acids. We investigated the effects of changes in light intensity on psbAII transcript levels in a series of light shift experiments in the wild-type Synechococcus sp. and in AMC084, a mutant which does not produce the 1.6-kb transcript. After exposure to high light intensities for 15 min, the level of the 1.2-kb psbAII transcript increased in both strains. This transcript was not detected in either strain after transfer to low light intensity. The psbAIII transcript showed the same pattern of response as the 1.2-kb psbAII transcript, whereas the 1.6-kb psbAII transcript was unaffected by different light intensities. The psbAI transcript levels responded oppositely to those of psbAII and psbAIII. These data, considered along with previous results obtained by using lacZ translational gene fusions, indicate that the response of psbA genes to changes in light intensity is controlled primarily at the transcriptional level.

Transcriptional and posttranscriptional components of psbA response to high light intensity in Synechococcus sp. strain PCC 7942.

The psbA genes, which encode the D1 protein of photosystem II, constitute a multigene family in the cyanobacterium Synechococcus sp. strain PCC 7942. Levels of messages from the three psbA genes change rapidly when cells are shifted from low-light to high-light conditions: the psbAI message level drops, whereas psbAII and psbAIII message levels increase dramatically. We examined the potential contributions of transcriptional and posttranscriptional processes in these high-light responses by subjecting cells that had been grown in a turbidostat at a standard light intensity (130 microeinsteins [microE] m-2 s-1) to either the same or a higher light intensity (500 microE m-2 s-1) in the presence or absence of rifampin. Northern (RNA blot) analysis of RNA isolated from cells subjected to high light showed that the increases in psbAII and psbAIII transcripts were blocked by rifampin. This suggests a transcriptional induction of these genes at high light intensities. Increased mRNA stability does not contribute to their accumulation in high-light conditions, since their half-life values did not increase relative to the half-lives measured at the standard light intensity. The rate of disappearance of the psbAI transcript in cells shifted to high light was diminished when either transcription or translation was blocked by rifampin or chloramphenicol, suggesting that accelerated degradation of the message requires de novo synthesis of a protein factor. When rifampin was added 10 min after the shift to high light intensity rather than before the shift, psbAI and psbAIII messages, but not the psbAII message, decayed at a faster rate. Susceptibility of the psbAIII transcript to the high-light-induced factor was also demonstrated by addition of chloramphenicol prior to the shaft to high light. psbAIII transcript levels went up more than twofold higher in chloramphenicol-treated cells than in untreated cells, whereas psbAII transcript levels were affected by the inhibitor. These experiments provide evidence that either new or increased synthesis of a degradation factor which affects a subset of Synechococcus transcripts occurs in cells subjected to high light intensity.

Complementation of a red-light-indifferent cyanobacterial mutant.

Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation.

Role of the Eikenella corrodens pilA locus in pilus function and phase variation.

The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.

Plasmids from two morphologically distinct cyanobacterial strains share a novel replication origin.

A 2.9-kbp replication origin from a plasmid endogenous to the filamentous cyanobacterium Fremyella diplosiphon UTEX 481 was genetically characterized and sequenced. Deletion analysis of the 2.9-kbp DNA fragment delimited the minimum region necessary for replication in F. diplosiphon Fd33 to approximately 2.5 kbp. DNA sequence analysis revealed that the F. diplosiphon plasmid replication origin is structurally very similar to and shares significant identity with the 1.75-kbp replication origin reported for plasmid pDU1, isolated from the morphologically distinct cyanobacterium Nostoc sp. strain PCC 7524. Each cyanobacterial plasmid replication origin includes a large open reading frame that predicts a conserved protein of unknown function; the predicted proteins of the replication origins are of similar sizes and 30% identical in amino acid sequence. Each cyanobacterial plasmid replication origin also possesses a region of dyad symmetry approximately 300 bp upstream of the conserved open reading frame.

A role for cpeYZ in cyanobacterial phycoerythrin biosynthesis.

Pigment mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon is characterized by constitutive synthesis of the phycobiliprotein phycoerythrin due to insertional inactivation of the rcaC regulatory gene by endogenous transposon Tn5469. Whereas the parental strain Fd33 harbors five genomic copies of Tn5469, cells of strain FdR1 harbor six genomic copies of the element; the sixth copy in FdR1 is localized to the rcaC gene. Electroporation of FdR1 cells yielded secondary pigment mutant strains FdR1E1 and FdR1E4, which identically exhibited the FdR1 phenotype with significantly reduced levels of phycoerythrin. In both FdR1E1 and FdR1E4, a seventh genomic copy of Tn5469 was localized to the cpeY gene of the sequenced but phenotypically uncharacterized cpeYZ gene set. This gene set is located downstream of the cpeBA operon which encodes the alpha and beta subunits of phycoerythrin. Complementation experiments correlated cpeYZ activity to the phenotype of strains FdR1E1 and FdR1E4. The predicted CpeY and CpeZ proteins share significant sequence identity with the products of homologous cpeY and cpeZ genes reported for Pseudanabaena sp. strain PCC 7409 and Synechococcus sp. strain WH 8020, both of which synthesize phycoerythrin. The CpeY and CpeZ proteins belong to a family of structurally related cyanobacterial proteins that includes the subunits of the CpcE/CpcF phycocyanin alpha-subunit lyase of Synechococcus sp. strain PCC 7002 and the subunits of the PecE/PecF phycoerythrocyanin alpha-subunit lyase of Anabaena sp. strain PCC 7120. Phycobilisomes isolated from mutant strains FdR1E1 and FdR1E4 contained equal amounts of chromophorylated alpha and beta subunits of phycoerythrin at 46% of the levels of the parental strain FdR1. These results suggest that the cpeYZ gene products function in phycoerythrin synthesis, possibly as a lyase involved in the attachment of phycoerythrobilin to the alpha or beta subunit.

Eikenella corrodens phase variation involves a posttranslational event in pilus formation.

The human pathogen Eikenella corrodens synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate E. corrodens VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designated pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin. The protein predicted by pilB shares sequence identity with the Dichelobacter nodosus FimB fimbrial assembly protein. The protein predicted by hagA resembles a hemagglutinin. The region containing these four ORFs was designated the pilA locus. DNA hybridization and sequence analysis showed that the pilA locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundant pilA1 transcript initiating upstream of pilA1 and terminating at a predicted hairpin structure between pilA1 and pilA2 was detected by several assays, as was a less abundant read-through transcript encompassing pilA1, pilA2, and pilB. Transcription from the pilA locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in E. corrodens.