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Nondestructive methods were used to evaluate marketed fiber-optic endoscopes (intended for simple viewing) for fluorescence recording. Our application is for optical recording from the heart. For one angioscope, we measured a focal length of 0.33 mm, a field of view of 45 degrees, an aperture of 0.26 mm, and an efficiency of 43%. We calculated that the angioscope would give a signal-to-noise ratio of 1.0 for a cardiac action potential, if its field of view were divided into a nine-pixel array (for safe continuous illumination). Our methods are useful in designing and evaluating fluorescence fiber-optic systems with superior signal quality and spatial resolution.

This study addresses the regulation of Staphylococcus aureus type 8 capsular polysaccharide (CP8) expression by the global regulators agr and sarA. We analyzed CP8 production, cap8-specific mRNA synthesis, and blaZ reporter gene activities of the transcriptional and translational fusions in strain Becker and its agr, sarA, and agr-sarA isogenic mutants during different phases of bacterial growth. In the wild-type strain, cap8 mRNA was undetectable until the mid-logarithmic phase of growth, whereas CP8 production was undetectable until 2 h later, at the onset of stationary phase. The delay most likely reflects the time needed for completing CP8 synthesis resulting from translation of cap8 mRNA. The agr mutation caused drastic reductions in CP8 production and cap8 gene transcription, suggesting that agr is a major positive regulator of CP8 expression. The results of gene fusion studies indicated that regulation by agr is exerted at the transcriptional level. In contrast, the sarA mutation caused only a slight reduction in cap8 mRNA synthesis and reporter gene activities. By comparing CP8 production and cap8 transcription, we observed that sarA affected CP8 production both trancriptionally and posttranslationally. We showed that agr was a major activator for cap gene expression not only in type 8 strain Becker but also in strains representing the four agr groups.

Retinal birefringence scanning (RBS) is a new technique that is used to detect the fixation of the eye remotely and noninvasively. The method is based on analysis of polarization changes induced by the retina. In this study, the principles of RBS were mathematically modeled to facilitate a better understanding of the origins of the signals obtained. Stokes vector analysis and Mueller matrix multiplication were augmented with Poincaré sphere representation. The cornea was modeled as a linear retarder. The foveal area was modeled as a radially symmetric birefringent medium. The model accurately predicted the frequency and phase of RBS signals obtained during central and paracentral fixation. The signal that indicates central fixation during RBS likely results from a combination of the radial birefringence of the Henle fibers and the overlying corneal birefringence.

The Staphylococcus aureus collagen adhesin (CNA) occurs in at least four forms that differ in the number (one, two, three, or four) of B domains. The B domains contain 187 amino acids and are located between the domains that anchor CNA to the cell envelope and the ligand-binding A domain. To determine whether a B domain is required for functional expression of CNA, we cloned the 2B cna gene from S. aureus strain Phillips and then eliminated both B domains by overlapping PCR. The absence of a B domain did not affect processing of the collagen adhesin to the cell surface or the ability to bind collagen. Based on our recent demonstration that the capsule can mask CNA on the surface of S. aureus cells (A. F. Gillaspy et al., Infect. Immun. 66:3170-3178, 1998), we also investigated the possibility that multiple B domains can extend the ligand-binding A domain outward from the cell surface and thereby overcome the inhibitory effect of the capsule. Specifically, we cloned the naturally occurring 4B CNA variant from S. aureus UAMS-639 and, by successive elimination of B domains, generated 1, 2, and 3B variants that are isogenic with respect to the 4B clone. After introducing each variant into microencapsulated and heavily encapsulated strains of S. aureus and growing cells under conditions known to affect capsule production (e.g., growth on Columbia agar), we correlated capsule production with exposure of CNA on the cell surface and the ability to bind collagen. Under no circumstance was the masking effect of the capsule reduced by the presence of multiple B domains. These results indicate that the B domains do not extend the ligand-binding A domain outward in a fashion that can overcome the inhibition of collagen binding associated with capsule production.

The production of type 8 capsular polysaccharide (CP8) in Staphylococcus aureus is regulated in response to a variety of environmental factors. The cap8 genes required for the CP8 production in strain Becker are transcribed as a single large transcript by a primary promoter located within a 0.45-kb region upstream of the first gene of the cap8 gene cluster. In this study, we analyzed the primary cap8 promoter region in detail. We determined the transcription initiation site of the primary transcript by primer extension and identified the potential promoter sequences. We found several inverted and direct repeats upstream of the promoter. Deletion analysis and site-directed mutagenesis showed that a 10-bp inverted repeat of one of the repeats was required for promoter activity. We showed that the distance but not the specific sequences between the inverted repeat and the promoter was critical to the promoter activity. However, insertion of a DNA sequence with two or four helix turns in this intervening region had a slight effect on promoter activity. To demonstrate the biological significance of the 10-bp inverted repeat, we constructed a strain with a mutation in the repeat in the S. aureus Becker chromosome and showed that the repeat affected CP8 production mostly at the transcriptional level. By gel mobility shift assay, we demonstrated that strain Becker produced at least one protein capable of specific binding to the 10-bp inverted repeat, indicating that the repeat serves as a positive regulatory protein binding site. In addition, reporter gene fusion analysis showed that the cap8 promoter activity was influenced by various growth media and affected most by yeast extract. Our results suggest that yeast extract may exert its profound inhibitory effect on cap8 gene expression through the 10-bp inverted repeat element.

To determine whether the ability of Staphylococcus aureus to bind collagen involves an adhesin other than the collagen adhesin encoded by cna, we examined the collagen binding capacity (CBC) of 32 strains of S. aureus. With only two exceptions, a high CBC corresponded with the presence of cna. Both exceptions involved cna-positive strains with a low CBC. The first was a single strain (ACH5) that encoded but did not express cna. The second were the mucoid strains Smith diffuse and M, both of which encoded and expressed cna but bound only minimal amounts of collagen. Analysis of capsule mutants suggests that the reduced CBC observed in the mucoid strains was due to masking of the collagen adhesin on the cell surface and that this masking effect is restricted to heavily encapsulated strains. Differences in the CBC of the remaining cna-positive strains were correlated to variations in the level of cna transcription and were independent of the number of B domain repeats in the cna gene. In all cna-positive strains other than ACH5, cna transcription was temporally regulated, with cna mRNA levels being highest in cells taken from exponentially growing cultures and falling to almost undetectable levels as cultures entered the post-exponential growth phase. The CBC was also highest with cells taken from exponentially growing cultures. Mutation of agr resulted in a slight increase in cna transcription and a corresponding increase in CBC during the exponential growth phase but did not affect the temporal pattern of cna transcription. Mutation of sar resulted in a more dramatic increase in CBC and a delay in the post-exponential-phase repression of cna transcription. Mutation of both sar and agr had an additive effect on both CBC and cna transcription. We conclude that (i) cna encodes the primary collagen-binding adhesin in S. aureus, (ii) sar is the primary regulatory element controlling expression of cna, and (iii) the regulatory effects of sar and agr on cna transcription are independent of the interaction between sar and agr.

The nucleotide sequences of two gene clusters, cap5 and cap8, involved in the synthesis of Staphylococcus aureus type 5 and type 8 capsular polysaccharides (CPs), respectively were determined. Each gene cluster contained 16 ORFs, which were named cap5A through cap5P for type 5 CP and cap8A through cap8P for type 8 CP. The cap5 and cap8 loci were allelic and were mapped to the SmaI-G fragment in the standard SmaI map of Staph. aureus strain NCTC 8325. The predicted gene products of cap5A through cap5G and cap5L through cap5P are essentially identical to those of cap8A through cap8G and cap8L through cap8P, respectively, with very few amino acid substitutions. Four ORFs located in the central region of each locus are type-specific. A comparison of the predicted amino acid sequences of cap5 and cap8 with sequences found in the databases allowed tentative assignment of functions to 15 of the 16 ORFs. The majority of the capsule genes are likely to be involved in amino sugar synthesis; the remainder are likely to be involved in sugar transfer, capsule chain-length regulation, polymerization and transport.

A 20.5-kb contiguous DNA fragment from Staphylococcus aureus Becker affecting type 8 capsule (CP8) biosynthesis was previously cloned. Sequencing analysis indicated that 16 open reading frames (ORFs) encoded within this fragment might be involved in CP8 synthesis. Using various plasmids containing DNA inserts derived from the 20.5-kb region, we showed by complementation of chemical mutants that 8 of the 16 ORFs were required for CP8 synthesis. To determine the involvement of the remaining eight ORFs, nonpolar gene-specific chromosomal mutations located in each of these ORFs were constructed. We found that three additional ORFs were also involved in the CP8 synthesis. Thus, 11 of the 16 ORFs were shown to affect CP8 synthesis. Complementation analyses of these 11 type 8 capsule (cap8) genes affecting CP8 production showed several promoters within the cap8 gene cluster. However, by Northern hybridization using either the entire cap8 gene cluster or the internal fragments of individual ORFs as probes, one 17-kb cap8-specific transcript was detected. Using xylE as the reporter gene, we found that the promoter at the beginning of the cap8 operon was much stronger than any of the internal promoters. These results suggest that the cap8 genes are transcribed mainly as a single large transcript. In addition, Southern hybridization analyses showed that cap8H, cap8I, cap8J, and cap8K, located in the central region of the cap8 gene cluster, were CP8 specific.

Eleven serotypes of capsular polysaccharide from Staphylococcus aureus have been reported. We have previously cloned a cluster of type 1 capsule (cap1) genes responsible for type 1 capsular polysaccharide biosynthesis in S. aureus M. To clone the type 8 capsule (cap8) genes, a plasmid library of type 8 strain Becker was screened with a labelled DNA fragment containing the cap1 genes under low-stringency conditions. One recombinant plasmid containing a 14-kb insert was chosen for further study and found to complement 14 of the 18 type 8 capsule-negative (Cap8-) mutants used in the study. Additional library screening, subcloning, and complementation experiments showed that all of the 18 Cap8- mutants were complemented by DNA fragments derived from a 20.5-kb contiguous region of the Becker chromosome. The mutants were mapped into six complementation groups, indicating that the cap8 genes are clustered. By Southern hybridization analyses under high-stringency conditions, we found that DNA fragments containing the cap8 gene cluster show extensive homology with all 17 strains tested, including type 1 strains. By further Southern analyses and cloning of the cap8-related homolog from strain M, we show that strain M carries an additional capsule gene cluster different from the cap1 gene cluster. In addition, by using DNA fragments containing different regions of the cap8 gene cluster as probes to hybridize DNA from different strains, we found that the central region of the cap8 gene cluster hybridizes only to DNAs from certain strains tested whereas the flanking regions hybridize to DNAs of all strains tested. Thus, the cap8 gene clusters and its closely related homologs are likely to have organizations similar to those of the encapsulation genes of other bacterial systems.

Forty temperature-sensitive mutations affecting lytic growth and eight affecting both establishment and maintenance of lysogeny of the temperate mycobacteriophage L1 have been isolated. All of the latter mutations form one complementation group and map within a very short region around the 15% coordinate of the L1 genome; these affect a single gene, cl, coding for the L1 repressor. The former 40 mutations form 28 complementation groups, identifying 28 different genes, G1-G28, essential for the lytic growth of L1. These genes have been mapped using the Gts mutations. Of the 28 Gts mutants, 14 are defective in host lysis at 42 degrees but not at 32 degrees while the other 14 can lyse the host at both temperatures. Among the former 14 Gts mutants, 6 are also defective in L1 DNA synthesis at 42 degrees, and they map in two different clusters, 4 around 65% and 2 around 84% of the L1 genome.