The acid-fastness of all mycobacteria is based upon a shared universal cell wall core structure. The mycobacterial cell wall consists of an outer lipid layer and an inner peptidoglycan layer. The outer layer is highly impermeable and is composed of unique 70 -90 carbon-containing lipids, known as mycolic acids. The mycolic acids are esterified to the non-reducing terminal arabinosyl residues of the polysaccharide arabinogalactan (1-5). The reducing end of arabinogalactan is connected to the peptidoglycan via the disaccharide linker, ␣-L-Rha-(133)␣-DGlcNAc-(13phosphate). Structural analyses showed that the integrity of the whole two-layer mycolic acid peptidoglycan assembly hinges on the presence of the rhamnosyl moiety as depicted in Fig. 1A. The complete structure of the linker is illustrated in Fig. 1B, and the reaction catalyzed by the enzyme, dTDP-Rha:␣-D-GlcNAc-pyrophosphate polyprenol, ␣-3-Lrhamnosyltransferase (referred to as rhamnosyltransferase in this study) is shown in Fig. 1C. The rhamnosyl residue and much if not all of the arabinogalactan polysaccharide are synthesized on GlcNAc-P-P-decaprenyl carrier lipid (6). The eventual transfer of the arabinogalactan-Rha-GlcNAc-phosphate unit to the O-6 of a muramic acid places the polysaccharide in mass onto the peptidoglycan. Finally, at some still to be defined point, the mycolic acids are attached to arabinofuranosyl residues at the non-reducing end of arabinogalactan.To further define and characterize the essential steps involved in the synthesis of the mycobacterial cell wall core, the classic microbial approach of isolating conditional lethal mutants was undertaken. Our strategy was to isolate temperature-sensitive (TS) 1 mutants in the genetically amenable and relatively fast growing Mycobacterium smegmatis mc 2 155 (7). A preferred large temperature range that would support growth precluded Mycobacterium tuberculosis from serving as the host for the generation of TS mutants. TS mutants would be genetically complemented with M. tuberculosis genomic DNA in hopes of identifying essential genes encoding cell wall biosynthetic enzymes. Herein, we describe the isolation of a TS cell wall mutant and the independent genetic complementation of that mutant with a M. tuberculosis gene and an E. coli gene. We report biochemical characterization of the TS mutant, the deduced amino acid change due to the mutation, the genetic complementation of an E. coli mutant to confirm the function of a M. tuberculosis gene, and the effect of the mutation on mycobacterial viability after exposure to non-permissive temperatures. EXPERIMENTAL PROCEDURESIsolation of TS Mutants-The strategy for the isolation and enrichment of bacterial TS mutants in a culture as outlined by A. Morris Hooke (8) was adapted for use in this study. M. smegmatis mc 2 155 (7) was inoculated into Middlebrook 7H9 with ADC supplement (Difco) (7H9) and grown at 37°C to ϳ10 8 colony-forming units/ml. Nitrosoguanidine (Sigma) was added to a final concentration of 0.1 mg/ml, and cultures were incubated at 37°C...

A 12.9 kb plasmid, pVT2, from a clinical Mycobacterium avium isolate, MD1, was cloned and radiolabeled for use as a DNA probe to examine the relatedness of plasmids in M. avium complex. That probe hybridized with plasmids isolated from M. avium complex strains from the environment (7 of 16) and from non-acquired immunodeficiency syndrome (AIDS) (10 of 17) and AIDS (5 of 6) clinical isolates. The similarity of plasmids from the environment with those from patients supports the hypothesis that the environment is a source of human M. avium complex infection. More striking was the observation that pVT2 hybridized with every plasmid (13 of 13 clinical and 5 of 5 environmental isolates) of 13.5 kb or smaller. A second probe, consisting of a 15.3 kb plasmid (pLR7) from another clinical isolate of the M. avium complex, hybridized with plasmids of 15.3 to 25 kb from environmental and clinical (AIDS and non-AIDS) isolates. There was no hybridization between pVT2 and pLR7. Thus, these two probes define two different groups of small mycobacterial plasmids.

Haemorrhagic enteritis virus (HEV) is a member of a genetically ill-defined group within the genus Aviadenovirus which causes significant clinical disease in gallinaceous fowl. Using DNA obtained from a low virulence isolate of HEV passed in turkeys, we developed a genomic restriction map and estimated an apparent genomic length of 25.5 kb. No evidence for extensive DNA hybridization was found between the HEV genome and either the hexon or penton base genes of human adenovirus 2 (HAdV-2) and fowl adenovirus 10 (FAdV-10). The HEV penton base gene was identified by PCR using primers based on conserved adenoviral DNA sequences. The penton base gene was expressed in Escherichia coli as a fusion protein and detected by anti-HEV serum in both colony and denaturing gel immunoblots. DNA sequencing revealed a putative penton base ORF with a predicted amino acid sequence showing approximately 39.0 %, 53.0 % and 44.2 % similarity with the penton base of HAdV-2, human adenovirus 40 (HAdV-40) and FAdV-10, respectively. The penton base gene was located at 43.3-48.6 m.u. on the HEV genome and had a remarkably low G + C content (33'8 %). DNA sequencing also revealed ORFs for putative core proteins resembling pVII, p-mu and a partial ORF similar to pVI (hexon-associated protein) of HAdV-2 and HAdV-40. The results support the claim that HEV represents a distinct group of viruses within the genus Aviadenovirus.

Assurance GDS(®) MPX ID for Top Shiga toxin-producing Escherichia coli (STEC; MPX ID) was validated according to the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Foods and Environmental Surfaces as (1) a secondary screening method for specific detection of the Top 6 STEC serogroups (O26, O45, O103, O111, O121, and O145) in raw beef trim, raw ground beef, raw spinach, and on stainless steel; and (2) as a confirmatory method for the identification of pure culture isolates as Top 6 STEC. MPX ID is used in conjunction with the upfront BCS Assurance GDS MPX Top 7 STEC assay. This Performance Tested Method(SM) validation has two main parts: Method Developer studies and the Independent Laboratory study. A total of 180 samples and controls were analyzed. Results showed that MPX ID had no statistically significant differences with the reference culture methods for the detection of Top 6 STEC in the food matrixes (raw beef trim, raw ground beef, and raw spinach) and environmental sponges (stainless steel) studied. Inclusivity/exclusivity studies were also conducted. One hundred percent inclusivity among the 50 Top 6 STEC serovars tested and 100% exclusivity for the 30 non-Top 6 STEC organisms tested were demonstrated. For validation of MPX ID as a confirmatory method for isolated colonies, all inclusivity and exclusivity organisms were streaked for isolation onto five STEC plating media: modified rainbow agar, Levine's eosin-methylene blue (L-EMB) agar, rainbow agar with novobiocin and cefixime, and enterohemolysin agar with selective agents as well as trypticase soy agar with yeast extract. These isolated colonies were suspended and analyzed by Assurance GDS MPX Top 7 STEC and MPX ID. MPX ID was able to correctly confirm all inclusivity organisms from all plate types, except two STEC isolates from L-EMB agar plates only in the Independent Laboratory study. All exclusivity organisms were correctly determined by MPX ID as non-Top 6 STEC from the STEC plating media. An additional but separate part of these studies was a comparison of immunomagnetic separation (IMS) efficiency using the Assurance GDS procedure with a PickPen(®) device and the U.S. Department of Agriculture procedure using the OctoMACS™ Separator device for plating onto chromogenic agar. Results demonstrated the equivalence of the two IMS procedures for plate confirmation of Top 7 STEC.

A multilaboratory collaborative study was conducted to compare the Assurance GDS™ for E. coli O157:H7 method and the reference culture methods for the detection of E. coli O157:H7 in orange juice, raw ground beef, and fresh lettuce. A separate companion assay, the Assurance GDS for Shigatoxin Genes method was also evaluated with the same test portions. Fifteen laboratories participated in the study. A Chi square analysis of each of the 3 food types at the high, low, and uninoculated control levels was performed. For all foods, the Assurance GDS for E. coli O157:H7 method and the Assurance GDS for Shigatoxin Genes method were equivalent to or better than the reference methods.