Examination of the Staphylococcus aureus nitric oxide reductase (saNOR) reveals its contribution to modulating intracellular NO levels and cellular respiration.

Staphylococcus aureus nitrosative stress resistance is due in part to flavohemoprotein (Hmp). Although hmp is present in all sequenced S. aureus genomes, 37% of analyzed strains also contain nor, encoding a predicted quinol-type nitric oxide (NO) reductase (saNOR). DAF-FM staining of NO-challenged wild-type, nor, hmp and nor hmp mutant biofilms suggested that Hmp may have a greater contribution to intracellular NO detoxification relative to saNOR. However, saNOR still had a significant impact on intracellular NO levels and complemented NO detoxification in a nor hmp mutant. When grown as NO-challenged static (low-oxygen) cultures, hmp and nor hmp mutants both experienced a delay in growth initiation, whereas the nor mutant's ability to initiate growth was comparable with the wild-type strain. However, saNOR contributed to cell respiration in this assay once growth had resumed, as determined by membrane potential and respiratory activity assays. Expression of nor was upregulated during low-oxygen growth and dependent on SrrAB, a two-component system that regulates expression of respiration and nitrosative stress resistance genes. High-level nor promoter activity was also detectable in a cell subpopulation near the biofilm substratum. These results suggest that saNOR contributes to NO-dependent respiration during nitrosative stress, possibly conferring an advantage to nor+ strains in vivo.

Arginine deiminase in Staphylococcus epidermidis functions to augment biofilm maturation through pH homeostasis.

Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.

Fatty acids and monoacylglycerols inhibit growth of Staphylococcus aureus.

Staphylococcus aureus causes a variety of human infections including toxic shock syndrome, osteomyelitis, and mastitis. Mastitis is a common disease in the dairy cow, and S. aureus has been found to be a major infectious organism causing mastitis. The objectives of this research were to determine which FA and esterified forms of FA were inhibitory to growth of S. aureus bacteria. FA as well as their mono-, di-, and triacylglycerol forms were tested for their ability to inhibit a human toxic shock syndrome clinical isolate (MN8) and two S. aureus clinical bovine mastitis isolates (305 and Novel). The seven most potent inhibitors across all strains tested by minimum inhibitory concentration analysis included lauric acid, glycerol monolaurate, capric acid, myristic acid, linoleic acid, cis-9, trans-11 conjugated linoleic acid, and trans-10, cis-12 conjugated linoleic acid. Some of these lipids were chosen for 48-h growth curve analysis with a bovine mastitis S. aureus isolate (Novel) at doses of 0, 20, 50, and 100 microg/mL except myristic acid, which was tested at 0, 50, 100, and 200 microg/mL. The saturated FA (lauric, capric, myristic) and glycerol monolaurate behaved similarly and reduced overall growth. In contrast, the polyunsaturated FA (linoleic and cis-9, trans-11 conjugated linoleic acid) delayed the time to initiation of exponential growth in a dose-dependent fashion. The results suggest that lipids may be important in the control of S. aureus during an infection.

The bactericidal action of penicillin: new clues to an unsolved mystery.

The Staphylococcus aureus lrgAB operon was recently shown to inhibit extracellular murein hydrolase activity and increase tolerance to penicillin. Further characterization of this operon could provide novel insight into the dynamics of S. aureus cell wall metabolism and the mechanism of penicillin-induced lethality.

The use of degenerate, sensor gene-specific, oligodeoxyribonucleotide primers to amplify DNA fragments from Staphylococcus aureus.

The sensor proteins of bacterial two-component regulatory systems comprise a large family of proteins that are involved in environmental sensing and signal transduction. To study these proteins in the Gram+ pathogen, Staphylococcus aureus, two pairs of degenerate oligodeoxyribonucleotides (oligos) that corresponded to conserved sequences contained within sensor protein-encoding genes were synthesized. Using these oligo primers, DNA fragments from S. aureus were amplified by polymerase chain reaction (PCR), cloned in Escherichia coli, and sequenced. Comparison of the deduced amino acid sequences from these cloned fragments to the sequences contained in the GenBank database suggest that some of the PCR products were derived from sensor protein-encoding genes. However, several other fragments were identified that encoded peptides with up to 65% identity to transport proteins. Given the biochemical and functional properties of some of these proteins, these data suggest that sensor and transport proteins may be evolutionarily related.

Analysis of Staphylococcus aureus genes encoding penicillin-binding protein 4 and an ABC-type transporter.

A region of the Staphylococcus aureus chromosome has been isolated that contains the gene encoding penicillin-binding protein 4 (PBP4), as well as abcA, a gene that encodes a protein with strong sequence similarity to the ABC transporter family of proteins. A disruption in abcA by Campbell-type integration results in cells that display an increased resistance to methicillin and cefoxitin, two antibiotics known to interact with low-molecular-weight PBPs. Based on these observations, a potential regulatory link between these two genes is discussed.

Identification and initial characterization of glucose-repressible promoters of Streptococcus mutans.

Three catabolite-repressible promoters from Streptococcus mutans have been isolated. These promoters were identified by utilizing the vector pRQ200 which contains a promoterless amylase-encoding gene, a Gram- origin of replication, and an erythromycin-resistance determinant. A library of S. mutans DNA was constructed in pRQ200, amplified in Escherichia coli and integrated by Campbell-type insertion into the S. mutans chromosome following transformation. Colonies exhibiting amylase production on media lacking an extraneous carbohydrate source were screened for diminished amylase production on media containing glucose. The effect of glucose on these promoters has been characterized using a quantitative spectrophotometric assay of amylase activity.

A genetic and molecular characterization of the recA gene from Staphylococcus aureus.

Previous studies have identified mutant strains of Staphylococcus aureus that have deficiencies in genetic recombination and DNA repair. Although these phenotypes were tentatively attributed to mutations within the S. aureus recA gene, experimental evidence to confirm this has never been reported. To characterize recA from S. aureus, we first isolated transposon insertion mutations that were in close proximity to the recA-like mutation (uvs-568) in strain 112 UVS-1. This allowed for the mobilization of the uvs-568 mutation into strain RN4220, the common laboratory strain of S. aureus. Next, using Bacillus subtilis recA as a probe, we cloned S. aureus recA and determined its nucleotide sequence. The deduced amino acid (aa) sequence of RecA contained 347 aa and was 74% identical to B. subtilis RecA. Using a cloned DNA fragment originating from within S. aureus recA, we then constructed a recA null mutant strain, designated KB103, which exhibited the same phenotypic characteristics imposed by the uvs-568 mutation in the same background. Furthermore, genetic and physical mapping of S. aureus recA placed it in the same region as the uvs-568 mutation. These data strongly suggest that these mutations represent different alleles of the same recA gene.

Age at onset of Alzheimer's disease. Relation to language dysfunction.

A later age at onset of Alzheimer's disease (AD) was found to be related to diminished language performance in 86 patients with probable AD. A hierarchical linear model was constructed to assess effects of age at onset and disease duration on the performance of patients with AD on four language tasks (naming, reading, auditory comprehension, and writing to dictation) after controlling for disease severity. Results of univariate analysis, in which the dependent variable was the averaged language task performances, revealed a significant effect for age at onset of AD, but not for disease duration. To assess the possibility that the relationship between the age at onset of AD and language performance reflects effects of normal aging, the language tasks were given to 33 normal subjects of similar ages who scored perfectly on dementia severity measures. A convincing relationship was not found between test score and age.

Change in cognitive function in idiopathic Parkinson disease.

OBJECTIVE: To study the effects of Parkinson disease (PD) on cognitive function by determining the frequency and amount of change in Mini-Mental State Examination (MMSE) performance. DESIGN: During a 4-year period, 77 patients with idiopathic PD and 43 normal elders were administered a neuropsychological test battery twice at 2 years apart. RESULTS: A 4-point score difference on the MMSE was the amount that was statistically calculated to be a significant difference at the .05 probability level. Using this metric, 17 (22%) patients with PD had a change in their MMSE performance during a 2-year period. Fifteen individuals performed poorer, and 2 individuals improved. Using the same metric, no normal subjects changed in their MMSE performance. The groups of patients with PD who had a change and did not have a change in their MMSE performance were not characterized by significant differences in their years of education, duration of illness, age at onset, age at test time 1, estimated premorbid intelligence, Hamilton Psychiatric Rating Scale for Depression score at test time I, or Unified Parkinson's Disease Rating Scale score. The singular difference was the higher frequency of change that was found in subjects who were taking dopamine agonists at the second test time. CONCLUSION: A change in cognitive function in patients with PD, as measured by a change of 4 points or more in their MMSE performance, was observed in only 22% of a sample of 77 patients with idiopathic PD during a 2-year period.

The recE(A)+ gene of B subtilis and its gene product: further characterization of this universal protein.

Although the SOS system of E coli and the SOB system of B subtilis share many similarities, there are distinct differences with respect to the regulation and specificity of the phenomena that constitute these global regulons. One of these differences resides in the regulation of the respective RecA and RecA-like proteins. In B subtilis the RecA-like protein, the RecE protein, shares 60% amino acid homology with its E coli counterpart. The E coli recA gene can complement most, but not all, of the functions that are lost in strains of B subtilis that do not produce a functional RecE protein. The DNA sequence of the recE+ gene as well as the sequence of the recE4 allele and the recA73 allele of B subtilis has demonstrated that mutants of the recE and recA loci of this bacterium actually represent alleles of the same complex gene. Accordingly, the major recombination protein of B subtilis should be referred to as RecA and the gene that encodes this protein as recA+.

Molecular characterization of regulatory elements controlling expression of the Bacillus subtilis recA+ gene.

Expression of the Bacillus subtilis recA gene is induced following DNA damage as well as during the development of the competent state. DNA damage-induction of the recA gene occurs by a RecA-dependent mechanism, whereas competence-induction occurs by a RecA-independent mechanism. To examine the molecular mechanisms that control the expression of the recA gene, a deletion analysis of the recA promoter region was performed. A regulatory region that is required for repression of recA expression was identified upstream of the recA promoter. Deletion of this regulatory region derepressed expression and abolished damage-induction of the recA promoter. Within this region are sequences similar to the consensus sequence that has been identified within DNA damage-inducible promoter regions of other B subtilis genes. Another regulatory region was identified that is required for the RecA-independent, competence-specific induction of the recA gene. Deletion of these sequences significantly reduced competence-induction of the recA promoter.

Comparing the difficulty of letter, semantic, and name fluency tasks for normal elderly and patients with Parkinson's disease.

Research on the effect of Parkinson's disease (PD) on verbal fluency has produced conflicting results. In this study, 88 PD patients with no dementia, 11 PD patients with questionable mental status, 15 PD patients with dementia, and 46 elders free from mental disorder were administered a variety of semantic, letter, and name fluency tasks. The results revealed that, contrary to popular assumption, semantic fluency was not always superior to letter fluency. Rather, verbal fluency was influenced by the nature of the individual categories. Interestingly, the relative difficulty of many categories was fairly stable across groups. The results also indicated that the individual fluency tasks were differentially sensitive to the mental status of the PD patients. Overall, the findings suggest that closer attention to the nature of the tested categories may help clarify the inconsistent effects of PD on verbal fluency.

Confrontation naming in Alzheimer's patients: relation to disease severity.

Reports of Alzheimer's disease patients in whom naming performance is disproportionate to other cognitive performances raise questions about the stage model, or dementia-severity level, for predicting naming performance. Thus, dementia severity as defined by Global Deterioration Scale ratings, Mini-Mental State Examination scores, and combinations of them was evaluated as a predictor of naming performance in 102 Alzheimer's patients and was found to account for approximately 1/3 of performance variability. Additional contributions from age at onset, duration, family history, and gender were negligible. Therefore, naming ability can be argued to have a subcomponent that is not subsumed by overall cognitive ability.

Caregiver report of prevalence and appearance order of linguistic symptoms in Alzheimer's patients.

We interviewed primary caregivers of 99 Alzheimer's disease patients about the existence and appearance order of linguistic symptoms in a longitudinal study of disease effects on communication. The most prevalent linguistic symptom was difficulty finding the correct word and the least prevalent was increased talkativeness. The prevalence of linguistic symptoms was strongly correlated with order of symptom appearance, with difficulty writing a meaningful letter appearing first and word finding difficulty appearing second. Based on caregiver perceptions, symptom prevalence and order of appearance are specified and discussed in relation to onset of nonlinguistic memory deficit, dementia severity, and performance on a linguistic communication test battery.

Confrontation naming impairment in dementia.

The effects of dementia etiology and severity on the confrontation naming ability of individuals with Alzheimer's, Huntington's, and Parkinson's diseases and multi-infarct dementia are investigated. Although naming impairment is reported as a consequence of dementing illness, confrontation naming is not found to be significantly impaired in mildly involved patients. Further, although moderate Huntington's and Parkinson's patients made more naming errors than normals, only moderate Alzheimer's disease patients are found to be significantly different. Regardless of etiology, most misnamings are found to be semantically related or semantically and visually related to the stimulus. Results challenge the theory that misnamings of dementia patients result primarily from misperception.

Response consistency on a semantic memory task in persons with dementia of the Alzheimer type.

The purpose of this investigation was to determine the test-retest response consistency rate on a semantic memory task in persons with dementia of the Alzheimer type (DAT). Ten mildly and 13 moderately impaired DAT subjects and 14 normal controls matched for age, years of education, and estimated IQ participated in this study. The Peabody Picture Vocabulary Test (PPVT) was administered twice to each subject with a 7-day intertest interval. The mild and moderate DAT subjects responded inconsistently to significantly more PPVT items than normal controls. When the effects of guessing were considered, moderate DAT subjects gave significantly more inconsistent PPVT responses than normal controls and mild DAT subjects showed a trend toward giving more inconsistent responses. These results substantiate the conclusion that the impairment of specific conceptual knowledge in DAT subjects cannot be reliably measured with a single administration of a semantic memory task such as the PPVT.

Naming and categorical knowledge in Alzheimer's disease: the process of semantic memory deterioration.

Semantic memory deterioration in Alzheimer's disease (AD) has been theorized to proceed from a loss of object attribute knowledge to a loss of category knowledge. The theory is based on the belief that naming is a computational process requiring object attribute knowledge. It is strengthened by reports that AD patients misname by giving category information and perform poorer on tests of attribute than category knowledge. The purpose of this study was to test the theory's validity by administering naming and category knowledge tasks to AD and normal elderly control subjects. Results revealed a theoretically unexpected outcome, that is, naming became easier relative to the recall and recognition of category information.

Phrase repetition in Alzheimer's disease: effect of meaning and length.

Repetition ability depends in part on the intactness of semantic memory. If the conceptual contents of semantic memory are lost as a function of Alzheimer's disease (AD) pathology, meaningfulness of stimuli should have progressively less effect on the ability to repeat as the disease worsens. This study was designed to evaluate the effects of meaningfulness and length of phrasal stimuli on repetition ability in mild and moderate AD patients and normal elderly subjects. Fifty-seven AD patients and 52 normal subjects were given six- and nine-syllable phrases that were meaningful, improbable in meaning, or meaningless. Cross-sectional and longitudinal data analyses were conducted and results failed to confirm a performance pattern consistent with a semantic memory loss theory. Meaningless nine-syllable phrases were those most difficult to repeat for moderate as well as mild AD patients and normal controls.

Relation of linguistic communication abilities of Alzheimer's patients to stage of disease.

A battery of linguistic communication (L-C) tasks was administered to 152 Alzheimer's disease patients in different stages of the disease and 60 normal elders. Subject performance data are used to construct a profile of L-C deficits by disease stage, as determined by ratings on the Global Deterioration Scale. Specification also is made of the L-C tasks on which mild Alzheimer's patients perform like normal elders, the relative difficulty of various L-C processes, the disease stage in which the greatest change occurs in L-C functions, and the degree of variation in L-C for individuals at a particular level of dementia severity.