Relationships between the history of thermal stress and the relative risk of diseases of Caribbean corals.

The putative increase in coral diseases in the Caribbean has led to extensive declines in coral populations. Coral diseases are a consequence of the complex interactions among the coral hosts, the pathogens, and the environment. Yet, the relative influence that each of these components has on the prevalence of coral diseases is unclear. Also unknown is the extent to which historical thermal-stress events have influenced the prevalence of contemporary coral diseases and the potential adjustment of coral populations to thermal stress. We used a Bayesian approach to test the hypothesis that in 2012 the relative risk of four signs of coral disease (white signs, dark spots, black bands, and yellow signs) differed at reef locations with different thermal histories. We undertook an extensive spatial study of coral diseases at four locations in the Caribbean region (10(3) km), two with and two without a history of frequent thermal anomalies (approximately 4-6 years) over the last 143 years (1870-2012). Locations that historically experienced frequent thermal anomalies had a significantly higher risk of corals displaying white signs, and had a lower risk of corals displaying dark spots, than locations that did not historically experience frequent thermal anomalies. By contrast, there was no relationship between the history of thermal stress and the relative risk of corals displaying black bands and yellow signs, at least at the spatial scale of our observations.

Caribbean yellow-band syndrome on Montastraea faveolata is not transmitted mechanically under field conditions.

Caribbean yellow-band syndrome is a highly prevalent coral disease whose transmission mechanisms are unknown. Affected corals often show multifocal lesions of yellow-colored tissue. We tested the hypothesis that a mechanical vector was responsible for these multifocal lesions. Four presumably non-resistant colonies were experimentally manipulated. Tissue and microbial assemblages were taken from diseased tissue and injected into healthy-looking tissue. Seawater injections were used as controls. The manipulations created a small wound, as would be generated by a coral predator. After 1 mo, all lesions healed and showed no signs of disease. We therefore reject the hypothesis that a mechanical vector, acting in a similar way as our manipulations, was responsible for the multifocal lesions.

Control of asgE expression during growth and development of Myxococcus xanthus.

One of the earliest events in the Myxococcus xanthus developmental cycle is production of an extracellular cell density signal called A-signal (or A-factor). Previously, we showed that cells carrying an insertion in the asgE gene fail to produce normal levels of this cell-cell signal. In this study we found that expression of asgE is growth phase regulated and developmentally regulated. Several lines of evidence indicate that asgE is cotranscribed with an upstream gene during development. Using primer extension analyses, we identified two 5' ends for this developmental transcript. The DNA sequence upstream of one 5' end has similarity to the promoter regions of several genes that are A-signal dependent, whereas sequences located upstream of the second 5' end show similarity to promoter elements identified for genes that are C-signal dependent. Consistent with this result is our finding that mutants failing to produce A-signal or C-signal are defective for developmental expression of asgE. In contrast to developing cells, the large majority of the asgE transcript found in vegetative cells appears to be monocistronic. This finding suggests that asgE uses different promoters for expression during vegetative growth and development. Growth phase regulation of asgE is abolished in a relA mutant, indicating that this vegetative promoter is induced by starvation. The data presented here, in combination with our previous results, indicate that the level of AsgE in vegetative cells is sufficient for this protein to carry out its function during development.

The asgE locus is required for cell-cell signalling during Myxococcus xanthus development.

In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell-cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor.

Screening for stimulant use in adult emergency department seizure patients.

OBJECTIVE: The objective of this study was to determine the prevalence of positive plasma drug screening for cocaine or amphetamine in adult emergency department seizure patients. METHODS: This prospective study evaluated consecutive eligible seizure patients who had a plasma sample collected as part of their clinical evaluation. Plasma was tested for amphetamine and the cocaine metabolite benzoylecgonine using enzyme-mediated immunoassay methodology. Plasma samples with benzoylecgonine greater than 150 ng/mL or an amphetamine greater than 500 ng/mL were defined as positive. Patient demographics, history of underlying drug or alcohol-related seizure disorder, estimated time from seizure to sample collection, history or suspicion of cocaine or amphetamine abuse, results of clinical urine testing for drugs of abuse, and assay results were recorded without patient identifiers. RESULTS: Fourteen of 248 (5.6%, 95% CI 2.7%-8.5%) plasma samples were positive by immunoassay testing for benzoylecgonine and no samples (0%, 95% CI 0-1.2%) were positive for amphetamine. Positive test results were more common in patient visits where there was a history or suspicion of cocaine or amphetamine abuse (p < 0.0005). CONCLUSIONS: During this study period, routine plasma screening for cocaine and amphetamines in adult seizure patients had a low yield. As a result, routine plasma screening would yield few cases of stimulant drug in which there was neither a history nor suspicion of drug abuse in this population.

SdeK is required for early fruiting body development in Myxococcus xanthus.

Myxococcus xanthus cells carrying the Omega4408 Tn5lac insertion at the sde locus show defects in fruiting body development and sporulation. Our analysis of sde expression patterns showed that this locus is induced early in the developmental program (0 to 2 h) and that expression increases approximately fivefold after 12 h of development. Further studies showed that expression of sde is induced as growing cells enter stationary phase, suggesting that activation of the sde locus is not limited to the developmental process. Because the peak levels of sde expression in both an sde+ and an sde mutant background were similar, we conclude that the sde locus is not autoregulated. Characterization of the sde locus by DNA sequence analysis indicated that the Omega4408 insertion occurred within the sdeK gene. Primer extension analyses localized the 5' end of sde transcript to a guanine nucleotide 307 bp upstream of the proposed start for the SdeK coding sequence. The DNA sequence in the -12 and -24 regions upstream of the sde transcriptional start site shows similarity to the sigma54 family of promoters. The results of complementation studies suggest that the defects in development and sporulation caused by the Omega4408 insertion are due to an inactivation of sdeK. The predicted amino acid sequence of SdeK was found to have similarity to the sequences of the histidine protein kinases of two-component regulatory systems. Based on our results, we propose that SdeK may be part of a signal transduction pathway required for the activation and propagation of the early developmental program.

Extragenic suppression of motA missense mutations of Escherichia coli.

The MotA and MotB proteins are thought to comprise elements of the stator component of the flagellar motor of Escherichia coli. In an effort to understand interactions among proteins within the motor, we attempted to identify extragenic suppressors of 31 dominant, plasmid-borne alleles of motA. Strains containing these mutations were either nonmotile or had severely impaired motility. Four of the mutants yielded extragenic suppressors mapping to the FlaII or FlaIIIB regions of the chromosome. Two types of suppression were observed. Suppression of one type (class I) probably results from increased expression of the chromosomal motB gene due to relief of polarity. Class I suppressors were partial deletions of Mu insertion sequences in the disrupted chromosomal motA gene. Class I suppression was mimicked by expressing the wild-type MotB protein from a second, compatible plasmid. Suppression of the other type (class II) was weaker, and it was not mimicked by overproduction of wild-type MotB protein. Class II suppressors were point mutations in the chromosomal motB or fliG genes. Among 14 independent class II suppressors characterized by DNA sequencing, we identified six different amino acid substitutions in MotB and one substitution in FliG. A number of the strongest class II suppressors had alterations of residues 136 to 138 of MotB. This particular region within the large, C-terminal periplasmic domain of MotB has previously not been associated with a specific function. We suggest that residues 136 to 138 of MotB may interact directly with the periplasmic face of MotA or help position the N-terminal membrane-spanning helix of MotB properly to interact with the membrane-spanning helices of the MotA proton channel.

Mutations in motB suppressible by changes in stator or rotor components of the bacterial flagellar motor.

Five proteins (MotA, MotB, FliG, FliM and FliN) may be involved in energizing flagellar rotation in Escherichia coli. To study interactions between the Mot proteins, and between them and the three Fli proteins of the switch-motor complex, we have isolated extragenic suppressors of dominant and partially dominant motB missense mutations. Four of the 13 motB mutations yielded partially allele-specific suppressors. Of the suppressing mutations, 57 are in the motA gene, eight are in fliG, and one is in fliM; no suppressor was identified in fliN. The prevalence of suppressors in fliG suggests that FliG interacts rather directly with the Mot proteins. The behaviour of cells in tethering and swarm assays indicates that the motA suppressors are more efficient than the fliG or fliM suppressors. Some of the suppressing mutations themselves confer distinctive phenotypes in motB+ cells. We propose a model in which mutations affecting residues in or near the putative peptidoglucan-binding region of MotB misalign the stator relative to the rotor. We suggest that most of the suppressors restore motility by introducing compensatory realignments in MotA or FliG.

Motility protein interactions in the bacterial flagellar motor.

Five proteins (MotA, MotB, FliG, FliM, and FliN) have been implicated in energizing flagellar rotation in Escherichia coli and Salmonella typhimurium. One model for flagellar function envisions that MotA and MotB comprise the stator of a rotary motor and that FliG, FliM, and FliN are part of the rotor. MotA probably functions as a transmembrane proton channel, and MotB has been proposed to anchor MotA to the peptidoglycan of the cell wall. To study interactions between the Mot proteins themselves and between them and other components of the flagellar motor, we attempted to isolate extragenic suppressors of 13 dominant or partially dominant motB missense mutations. Four of these yielded suppressors, which exhibited widely varying efficiencies of suppression. The pattern of suppression was partially alleles-specific, but no suppressor seriously impaired motility in a motB+ strain. Of 20 suppressors from the original selection, 15 were characterized by DNA sequencing. Fourteen of these cause single amino acid changes in MotA. Thirteen alter residues in, or directly adjacent to, the putative periplasmic loops of MotA, and the remaining one alters a residue in the middle of the fourth predicted transmembrane helix of MotA. We conclude that the MotA and MotB proteins form a complex and that their interaction directly involves or is strongly influenced by the periplasmic loops of MotA. The 15th suppressor from the original selection and 2 motB suppressors identified during a subsequent search cause single amino acid substitutions in FliG. This finding suggests that the postulated Mot-protein complex may be in close proximity to FliG at the stator-rotor interface of the flagellar motor.