Oxygen intervention in the regulation of gene expression: the photosynthetic bacterial paradigm.

The means by which oxygen intervenes in gene expression has been examined in considerable detail in the metabolically versatile bacterium Rhodobacter sphaeroides. Three regulatory systems are now known in this organism, which are used singly and in combination to modulate genes in response to changing oxygen availability. The outcome of these regulatory events is that the molecular machinery is present for the cell to obtain energy by means that are best suited to prevailing conditions, while at the same time maintaining cellular redox balance. Here, we explore the dangers associated with molecular oxygen relative to the various metabolisms used by R. sphaeroides, and then present the most recent findings regarding the features and operation of each of the three regulatory systems which collectively mediate oxygen control in this organism.

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: effect of a transposon insertion in the hbdA gene.

The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-beta-hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.

The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 is an alpha-3 purple nonsulfur eubacterium with an extensive metabolic repertoire. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. Photosynthesis may be photoheterotrophic using organic compounds as both a carbon and a reducing source, or photoautotrophic using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. The structural components of this metabolically diverse organism and their modes of integrated regulation are encoded by a genome of approximately 4.5 Mb in size. The genome comprises two chromosomes CI and CII (2.9 and 0.9 Mb, respectively) and five other replicons. Sequencing of the genome has been carried out by two groups, the Joint Genome Institute, which carried out shotgun-sequencing of the entire genome and The University of Texas-Houston Medical School, which carried out a targeted sequencing strategy of CII. Here we describe our current understanding of the genome when data from both of these groups are combined. Previous work had suggested that the two chromosomes are equal partners sharing responsibilities for fundamental cellular processes. This view has been reinforced by our preliminary analysis of the virtually completed genome sequence. We also have some evidence to suggest that two of the plasmids, pRS241a and pRS241b encode chromosomal type functions and their role may be more than that of accessory elements, perhaps representing replicons in a transition state.

Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli.

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1. In common with other ALASs the recombinant R. sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.

Preliminary investigation of voice onset time production in persons with dysphagia.

The purpose of this study was to determine whether voice onset time (VOT) values of persons with dysphagia differed from those of a person with normal swallow function. Five male subjects with dysphagia (average age = 80.6 years) and a control subject (age = 79 years) read 18 consonant-vowel-consonant words in quasi-random order. These syllables began with the voiced and voiceless cognates from the three stop places of articulation (i.e., bilabial, alveolar, and velar). These consonants were followed by the vowels /i/, /a/, and /u/. Digital audio tape recordings were performed and speech was digitized onto disk. Measurements were completed using BLISS software (Mertus J: BLISS User's Manual. Providence: Department of Cognitive and Linguistic Sciences, Brown University, 1989) implemented on a 486 microcomputer. Averages and standard deviations of the VOT measures for the six stop consonants were compared between the two experimental groups. For the dysphagic speakers, average VOT values for voiceless stops were shorter, and there were larger negative VOT values for voiced stops. Standard deviations for the VOT productions pf the dysphagic subjects were smaller. Statistical comparisons showed significant differences between individual dysphagic speakers and the normal control for three of the five subjects. These preliminary data suggest that dysphagia affects the fine motor control required for accurate VOT production in speech.

Voice onset time in Spanish-English bilinguals: early versus late learners of English.

Thirty-two Hispanic speakers of English were evenly divided into two groups based on whether or not their initial learning of English began prior to, or after the age of 12 years. Each group had an even number of males (16) and females (16). The subjects were recorded producing a protocol of 18 basic speech syllables. The first three repetitions (54 tokens) were chosen for analysis. The 1728 tokens were digitized and measured for voice onset time (VOT). Findings support the hypothesis that the VOT values of Hispanics speaking English differ according to whether initial learning of English began prior to or after the age of 12 years. An analysis of variance (ANOVA) found significant main effects of group, place, voice, and gender. Significant interactions were group by voice, and voice by gender.

Role of the fnrL gene in photosystem gene expression and photosynthetic growth of Rhodobacter sphaeroides 2.4.1.

Anoxygenic photosynthetic growth of Rhodobacter sphaeroides 2.4.1 requires a functional fnrL gene, which encodes the anaerobic regulator, FnrL. Using transcriptional fusions to the puc operon in which the upstream FNR consensus-like sequence is either present or absent, we obtained results that suggest that FnrL has both a direct and an indirect role in puc operon expression. In addition to FnrL, several other factors, including the two-component Prr regulatory system and the transcriptional repressor PpsR, are known to mediate oxygen control of photosynthesis gene expression in this organism. Therefore, we examined the relationship between FnrL and these other regulatory elements. Our results indicate that while mutations of prr or ppsR can lead to an increase in expression of some photosynthesis genes under aerobic and anaerobic conditions, regardless of the presence or absence of FnrL, there remains an absolute requirement for a functional fnrL gene for photosynthetic growth. We examined the potential role(s) of FnrL in photosynthetic growth by considering several target genes which may be required for this growth mode.

A comparison of the prosodic characteristics of the speech of people with Parkinson's disease and Friedreich's ataxia with neurologically normal speakers.

The realization of prosody (speech rate, fundamental frequency, intonation) was investigated in a group of 10 individuals with Parkinson's disease and a group of 10 individuals with Friedreich's ataxia. Data from these two neurologically disordered groups were compared to individuals without neurological impairment. Both neurologically impaired groups retained some aspects of normal speech prosody, while other aspects were affected to a significant degree. The prosodic characteristics of speakers with Parkinson's disease were distinct from those of speakers with Friedreich's ataxia. These results were interpreted in terms of prosodic competence and prosodic performance.

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.

A preliminary investigation of the effects of gender and race on Voice Onset Time.

Twenty individuals participated in a study of Voice Onset Time (VOT) production. Participants included equal numbers of males and females and equal numbers of African Americans and Caucasian Americans. Each individual read a set of stimuli formed from the six stop consonants (/p/,/t/,/k/;/b/,/d/,/g/) combined with the three vowels /i/,/a/, and /u/. Their productions were measured for VOT. Considerably more prevoicing (i.e., negative VOT) for voiced stops was found in the present study in comparison with past studies. Statistically significant differences were found for both gender and race. These results suggest that the normative data presently available is probably inadequate because it does not accurately reflect the normal distribution of either gender or race within the American population.

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state.

Rhodobacter sphaeroides 2.4.1 has the ability to synthesize a variety of tetrapyrroles, reflecting the metabolic versatility of this organism and making it capable of aerobic, anaerobic, photosynthetic, and diazotrophic growth. The hemA and hemT genes encode isozymes that catalyze the formation of 5-aminolevulinic acid, the first step in the biosynthesis of all tetrapyrroles present in R. sphaeroides 2.4.1. As part of our studies of the regulation and expression of these genes, we developed a genetic selection that uses transposon mutagenesis to identify loci affecting the aerobic expression of the hemA gene. In developing this selection, we found that sequences constituting an open reading frame immediately upstream of hemA positively affect hemA transcription. Using a transposon-based selection for increased hemA expression in the absence of the upstream open reading frame, we isolated three independent mutants. We have determined that the transposon insertions in these strains map to three different loci located on chromosome 1. One of the transposition sites mapped in the vicinity of the recently identified R. sphaeroides 2.4.1 homolog of the anaerobic regulatory gene fnr. By marker rescue and DNA sequence analysis, we found that the transposition site was located between the first two genes of the cco operon in R. sphaeroides 2.4.1, which encodes a cytochrome c terminal oxidase. Examination of the phenotype of the mutant strain revealed that, in addition to increased aerobic expression of hemA, the transposition event also conferred an oxygen-insensitive development of the photosynthetic membranes. We propose that the insertion of the transposon in cells grown in the presence of high oxygen levels has led to the generation of a cellular redox state resembling either reduced oxygen or anaerobiosis, thereby resulting in increased expression of hemA, as well as the accumulation of spectral complex formation. Several models are presented to explain these findings.

In vivo protein folding: suppressor analysis of mutations in the groES cochaperone gene of Escherichia coli.

Our previous work has shown that the Escherichia coli groES14 and groES15 mutations result in reduced GroE chaperone machine function. By selecting for restoration of the ability of those mutant groES alleles to suppress the thermosensitivity of bacteria bearing the dnaA46 mutation, we isolated a number of intra- and extragenic suppressors that increase in vivo GroE chaperone function. One of the intragenic suppressors has been mapped to a segment that codes for the GroES mobile loop, previously shown to be indispensable for proper GroES/GroEL interaction. Two extragenic suppressors have been mapped to a groEL segment, previously identified by mutational analysis as coding for an important functional region of the GroEL protein. Our results should contribute to our eventual understanding of the structure-function relationships of the universally conserved GroE chaperone machine.

Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene.

In Rhodobacter sphaeroides 2.4.1, the cellular requirements for 5-aminolevulinic acid (ALA) are in part regulated by the level of ALA synthase activity, which is encoded by the hemA and hemT genes. Under standard growth conditions, only the hemA gene is transcribed, and the level of ALA synthase activity varies in response to oxygen tension. The presence of an FNR consensus sequence upstream of hemA suggested that oxygen regulation of hemA expression could be mediated, in part, through a homolog of the fnr gene. Two independent studies, one detailed here, identified a region of the R. sphaeroides 2.4.1 genome containing extensive homology to the fix region of the symbiotic nitrogen-fixing bacteria Rhizobium meliloti and Bradyrhizobium japonicum. Within this region that maps to 443 kbp on chromsome I, we have identified an fnr homolog (fnrL), as well as a gene that codes for an anaerobic coproporphyrinogen III oxidase, the second such gene identified in this organism. We also present an analysis of the role of fnrL in the physiology of R. sphaeroides 2.4.1 through the construction and characterization of fnrL-null strains. Our results further show that fnrL is essential for both photosynthetic and anaerobic-dark growth with dimethyl sulfoxide. Analysis of hemA expression, with hemA::lacZ transcriptional fusions, suggests that FnrL is an activator of hemA under anaerobic conditions. On the other hand, the open reading frame immediately upstream of hemA appears to be an activator of hemA transcription regardless of either the presence or the absence of oxygen or FnrL. Given the lack of hemT expression under these conditions, we consider FnrL regulation of hemA expression to be a major factor in bringing about changes in the level of ALA synthase activity in response to changes in oxygen tension.

Voice onset time production in French-speaking aphasics.

VOT productions were compared for 5 Broca's aphasics and 5 Wernicke's aphasics to those for a group of 5 normal subjects of similar age. Phonetic errors were produced by both types of aphasic patients, but not by normal controls. Although previous studies have found significantly more phonetic errors in VOT production for Broca's aphasics, this study did not. Both Broca aphasics and Wernicke aphasics had somewhat less overall average difference in VOT between voiced and voiceless consonant pairs than normal speakers. Standard deviations associated with the VOT productions were also greater for both aphasic groups than for normal subjects. The results of this study are considered in light of the previous literature.

Regulation of 5-aminolevulinic acid synthesis in Rhodobacter sphaeroides 2.4.1: the genetic basis of mutant H-5 auxotrophy.

Rhodobacter sphaeroides H-5 was isolated as a 5-aminolevulinic acid (ALA) auxotroph following treatment of wild-type cells with N-methyl-N-nitroso-N'-nitroguanidine (J. Lascelles and T. Altshuler, J. Bacteriol. 98:721-727, 1969). The existence in R. sphaeroides 2.4.1 of the genes hemA and hemT, each encoding the enzyme 5-aminolevulinic acid synthase (EC 2.3.1.37), raised questions as to the genetic basis for the ALA auxotrophy in mutant H-5. We therefore cloned both the hemA and hemT genes from mutant H-5. The hemA gene has been sequenced in its entirety and bears four base pair substitutions which encode three amino acid changes relative to the sequence of wild-type strain 2.4.1. Complementation analysis of an Escherichia coli ALA auxotroph has revealed that the loss of ALA synthase activity in the HemA mutant enzyme could be localized to two of the amino acid substitutions. On the other hand, the hemT gene from mutant H-5 was able to complement an E. coli mutant requiring ALA for growth. Complementation analyses were also carried out by introducing the cloned hemA or hemT gene of mutant H-5 or wild-type 2.4.1 in trans into H-5 and, in parallel, into our previously described HemA-HemT double mutant strain AT1 (E. L. Neidle and S. Kaplan, J. Bacteriol. 175:2304-2313, 1993). This analysis revealed that while the complementation pattern of mutant AT1 parallels that for the E. coli ALA auxotroph, mutant H-5 could only be complemented by the wild-type hemA gene. The ability of the hemT gene of either mutant H-5 or wild-type 2.4.1 to complement the ALA auxotrophy of mutant AT1 but not mutant H-5 was consistent with beta-galactosidase activities obtained with hemT-lacZ transcriptional fusions. We conclude that the ALA auxotrophy of mutant H-5 arises from (i) a nonfunctional HemA protein containing multiple missense substitutions and (ii) an inability of the normal hemT gene to be expressed in the mutant H-5 genetic background, i.e., an additional mutation of unknown origin is required for hemT expression. These studies bear directly on the regulation of the expression of the hemA and hemT genes of R. sphaeroides 2.4.1.

Improving fundamental frequency modulation in head trauma patients: a preliminary comparison of speech-language therapy conducted with and without IBM's Speech Viewer.

This study involves a preliminary comparison of traditional speech therapy for head trauma patients with therapy assisted by computer. Two subjects were treated in a 'crossover' design to compare the effects of each treatment. While both subjects improved, there was no obvious difference in treatment effectiveness. However, the computer support provided the measures of treatment efficacy which made this comparison possible.

[Values of certain prosodic parameters obtain with French-speaking probands without communication problems]. / Valeurs de certains paramètres prosodiques obtenues auprès de sujets francophones sans trouble de la communication.

Acoustic data are provided in order to facilitate evaluation of the speech of adult patients with prosody disorders such as dysarthria. Forty French-speaking subjects between 45 and 75 years of age, with neither neurological disease nor a communication disorder, were grouped on the basis of sex and age. Subjects were required to produce four series of 20 matched interrogative and declarative sentences. Their productions were recorded and analyzed with an IBM Speech Viewer. Measures of fundamental frequency, of variation in fundamental frequency, of rate over the entire sentence, as well as a measure of intonation (defined as the difference in fundamental frequency between the last syllable of matched declarative and interrogative sentences) are reported in tables for clinical use. These data provide information for the evaluation of the prosody of French-speaking persons.