Nonreplicating persistence of mycobacterium tuberculosis.

There is ample clinical evidence, as well as evidence from animal experiments, that Mycobacterium tuberculosis can persist in tissues for months to decades without replicating, yet with the ability to resume growth and activate disease. Our knowledge of both macrophage physiology and the nature of tuberculous lesions in man and animals suggests that hypoxia is a major factor in inducing nonreplicating persistence (NRP) of tubercle bacilli. In vitro models reinforce this conclusion and provide insights into mechanisms that make NRP possible. There is evidence from in vitro models that the strategies employed by the bacilli to permit hypoxic NRP include restriction of biosynthetic activity to conserve energy, induction of alternative energy pathways, and stabilization of essential cell components to lessen the need for repair or replacement.

Microaerophilic induction of the alpha-crystallin chaperone protein homologue (hspX) mRNA of Mycobacterium tuberculosis.

Among the products that are expressed when Mycobacterium tuberculosis undergoes hypoxic shiftdown to nonreplicating persistence (NRP) is the alpha-crystallin chaperone protein homologue (Acr). This expression coincides with the previously reported appearance of a respiratory type of nitrate reductase activity, the increase in glycine dehydrogenase activity, and the production of a unique antigen, URB-1. In a timed sampling study, using a slowly stirred oxygen depletion culture model, we have demonstrated that the hspX mRNA that codes for Acr protein as well as the protein itself is induced just as the bacilli enter the microaerophilic NRP stage 1 (NRP-1). In contrast to the induction observed for hspX mRNA, levels of 16S rRNA, fbpB mRNA (encoding the 85B alpha antigen), and aroB mRNA (encoding dehydroquinate synthase) demonstrate relatively small to no change upon entering NRP-1. Acr protein was shown to be identical to URB-1 by Western analysis with anti-URB-1 antibody. The fact that antibody to Acr is found in a high percentage of tuberculosis patients suggests that the hypoxic shiftdown of tubercle bacilli to the NRP state that occurs in vitro, resulting in production of the alpha-crystallin protein, occurs in vivo as well. Simultaneous abrupt increases in hspX mRNA and Acr protein suggest that Acr protein expression is controlled at the level of transcription.

Surface protein variation by expression site switching in the relapsing fever agent Borrelia hermsii.

Borrelia hermsii, an agent of relapsing fever, undergoes antigenic variation of serotype-specifying membrane proteins during mammalian infections. When B. hermsii is cultivated in broth medium, one serotype, 33, eventually predominates in the population. Serotype 33 has also been found to be dominant in ticks but not in mammalian hosts. We investigated the biology and genetics of two independently derived clonal populations of serotype 33 of B. hermsii. Both isolates infected immunodeficient mice, but serotype 33 cells were limited in number and were only transiently present in the blood. Probes for vsp33, which encodes the serotype-specifying Vsp33 outer membrane protein, revealed that the gene was located on a 53-kb linear plasmid and that there was only one locus for the gene in serotype 33. The vsp33 probe and probes for other variable membrane protein genes showed that expression of Vsp33 was determined at the level of transcription and that when the vsp33 expression site was active, an expression site for other variable proteins was silent. The study confirmed that serotype 33 is distinct from other serotypes of B. hermsii in its biology and demonstrated that B. hermsii can change its major surface protein through switching between two expression sites.

Spirochaeta aurantia has diacetyl chloramphenicol esterase activity.

The free-living spirochete Spirochaeta aurantia was nearly as susceptible to diacetyl chloramphenicol, the product of chloramphenicol acetyltransferase, as it was to chloramphenicol itself. This unexpected susceptibility to diacetyl chloramphenicol was wholly or partly the consequence of intrinsic carboxylesterase activity, as indicated by high-performance liquid chromatography, thin-layer chromatography, and microbiological assays. The esterase converted the diacetate to chloramphenicol, thus inhibiting spirochete growth. The esterase activity was cell associated, reduced by proteinase K, eliminated by boiling, and independent of the presence of either chloramphenicol or diacetyl chloramphenicol. S. aurantia extracts also hydrolyzed other esterase substrates, and two of these, alpha-napthyl acetate and 4-methylumbelliferyl acetate, identified an esterase of approximately 75 kDa in a nondenaturing gel. Carboxylesterases occur in Streptomyces species, but in this study their activity was weaker than that of S. aurantia. The S. aurantia esterase could reduce the effectiveness of cat as either a selectable marker or a reporter gene in this species.

The extended promoters for two outer membrane lipoprotein genes of Borrelia spp. uniquely include a T-rich region.

OspA and B proteins of Borrelia burgdorferi and Vmp proteins of Borrelia hermsii are abundant outer membrane lipoproteins, whose expression varies with the environment. The genes for these proteins have the '-35' and '-10' elements of a sigma70-type promoter. Deletions of the promoters for these genes were analysed with a chloramphenicol acetyltransferase (CAT) reporter gene and plasmid constructs that were stably maintained in Escherichia coli or transiently transfected into B. burgdorferi. Reporter expression was measured as susceptibility of transformed E. coli cells to chloramphenicol and the CAT activity of E. coli and B. burgdorferi lysates in vitro. Presence of the '-10' element was essential for full activity in both B. burgdorferi and E. coli. Upstream of the '-35' elements of the ospAB and vmp promoters were tracts with Ts in 16 of 20 positions for B. burgdorferi and 18 of 20 positions for B. hermsii. Deletion of the T-rich region from the ospAB or vmp promoter caused a greater reduction of CAT activity in B. burgdorferi than in E. coli. The findings indicate that ospAB and vmp promoters are extended promoters with two parts: (i) a core region containing typical '-35' and '-10' elements and (ii) a unique T-rich region.

Esterases in serum-containing growth media counteract chloramphenicol acetyltransferase activity in vitro.

The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that of B. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility of E. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. coli bearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo.

Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.

Tn4556 and luciferase: synergistic tools for visualizing transcription in Streptomyces.

Progress in understanding genetic regulatory controls in the Actinomycetes has been rate limited by the properties of in vivo transcriptional probes. We have developed a set of plasmid- and transposon-based promoter-probe vectors that employ the Vibrio harveyi luciferase-encoding luxAB cassette as a reporter of transcription. The primary advantages of luciferase (Lux) over other reporter gene products are: (i) unsurpassed sensitivity; (ii) utility during stationary-phase gene expression; and (iii) the ability to localize promoter activity spatially within developing colonies. We have used these vectors to screen for Streptomyces coelicolor promoters that exhibit developmental phenotypes or that are induced by various environmental stimuli. The plasmid-based probes have proved invaluable for identifying cis- and trans-acting elements that are required for stationary-phase expression of the S. coelicolor sapA gene. A collection of novel bld and whi insertion mutants has been obtained by use of the Lux-encoding transposon, Tn5353.

Construction and application of plasmid- and transposon-based promoter-probe vectors for Streptomyces spp. that employ a Vibrio harveyi luciferase reporter cassette.

Several versatile promoter-probe vectors have been constructed for Streptomyces strains which utilize the production of blue-green light as a measure of transcription activity. Three plasmid vectors (two high and one low copy number) and two transposons are described. The multicopy plasmids pRS1106 and pRS1108 contain a transcription terminator and multiple-cloning polylinker upstream of promoterless luciferase (lux) and neomycin resistance reporter genes. Plasmid pHI90 is similar in structure to the pRS vectors except that its single copy number is an advantage for regulation studies or situations in which overexpression is otherwise toxic to the cell. The two transposons carry a promoterless lux cassette cloned such that transposition into a target DNA and fusion to the target's transcription unit occur simultaneously. Tn5351 was created by inserting the luciferase genes near the right end of the viomycin resistance transposon Tn4563. Tn5353 carries the luciferase genes near the right end of a neomycin resistance transposon derived from Tn4556. The size of Tn5353 was minimized by deleting nonessential transposon sequences, making this element small enough to be cloned into phi C31 bacteriophages for efficient transposon delivery to target cells of Streptomyces strains. The two Tnlux transposons have been used to generate Streptomyces coelicolor morphological mutants and to monitor transcription from chromosomal promoters during development.