MELLING. 1992. Gram-positive bacilli isolated during epidemiological investigations which, on the basis of conventional tests, resemble Bacillus anthracis but which fail to produce the capsule or to induce anthrax in test animals have long been dismissed in clinical and veterinary laboratories as B . cereus or simply as unidentified Bacillus spp. and thereupon discarded as inconsequential. In this study, the application of newly available DNA probe, polymerase chain reaction and specific toxin antigen detection technology has revealed that a proportion of such strains are B . anthracis which lack the plasmid carrying the capsule gene (pX02). While these techniques cannot, of course, be used to confirm the identities of strains resembling B . anthracis but which also lack the plasmid carrying the toxin genes (pXOl), the likelihood that these also are bonaJde B . anthracis becomes more acceptable. (As yet no naturally occurring pXOl-/2+ strains have been found.) At this point, the significance of the presence of such avirulent forms of B. anthracis in specimens can only be a subject for speculation, but the possibility that they may be indicators of virulent parents somewhere in the system being examined must be considered.

1. Pseudomonas arvilla mt-2 grows a t the expense of benzoate, m-toluate (3-methylbenzoate) and p-toluate (4-methylbenzoate), but not o-toluate (2-methylbenzoate). Under various conditions compounds with spectra characteristic of muconic semialdehydes can be made to accumulate in the media. These spectra indicate that benzoate is metabolised through catechol and 2-hydroxymuconic semialdehyde (2-hydroxy-6-oxohexa-2,4-dienoate), m-toluate through 3-methylcatechol and 2-hydroxy-6-oxohepta-2,4-dienoate and p-toluate through 4-methylcatechol and 2-hydroxy-5-methylmuconic semialdehyde (2-hydroxy-5-methyl-6-oxohexa-2,4-dienoate).2. Freshly harvested cells grown on all three substrates only consume oxygen a t a significant rate when presented with the growth substrates themselves and catechol and the methylcatechols, but not with salicylate, protocatechuate or phenol or the cresols.3. Extracts of cells grown on these substrates contain high induced levels of the suite of meta cleavage enzymes, including both the hydrolytic branch and the 4-oxalocrotonate branch.4. The substrate specificity of the early enzymes of the pathway suggests that only one nonspecific enzyme expresses each activity in cell-free extracts and that it is nonspecifically induced during growth on all three carbon sources. 5.It is suggested that the metabolic role of the hydrolytic branch of the pathway is the assimilation of 3-methylcatechol and its precursor, and that of the 4-oxalocrotonate branch is to assimilate catechol and 4-methylcatechol and their precursors.6. o-Toluate is not metabolised because it is neither a substrate for the benzoate oxidase system nor an inducer of any of the meta pathway enzymes.7 . It appears that benzoate and m-and p-toluates are the substrate inducers of the meta pathway enzymes in this organism. The ortho pathway is induced on incubating cells with catechol and the first inducer appears to be &,cis-muconate or possibly catechol itself.The coexistence of two enzymes able to degrade the meta cleavage product of catechol, 2-hydroxymuconic semialdehyde, was first demonstrated in cell-free extracts of both benzoate-grown Azotobacter species [l] and a naphthalene-grown Pseudomonas strain NCIB 9816 [2]. Sala-Trepat and Evans [3] elucidated the two resulting pathways, which after diverging a t 2-hydroxymuconic semialdehyde converge again a t 2-oxopent-4-enoate ; they concluded that in Azotobacter only the 4-oxalocrotonate branch, involving as the first step the NADf-dependent dehydrogenation of 2-hydroxymuconic semialdehyde, was of any metabolic significance since the 2-hydroxymuconic semialdehyde hydrolase was present Enzymes. Catechol 1,2-oxygenase or catechol: oxygen 1,2-oxidoreductase (EC 1.13.1.1); catechol 2,3-oxygenase or catecho1:oxygen 2,3-oxidoredoctase (EC 1.13.1.2); NAD nucleosidase or NAD glycohydrolase (EC 3.2.2.6). 21 Eur. J. Blochem., Vol.28 in only very low uninducible levels. I n NCIB 9816 all the enzymes of the 4-oxalocrotonate branch are present [4] and again it has been proposed that catechol is metab...

Some of a set of independently arising Tol- (non toluate-utilising) derivatives of Pseudomonas putida mt-2 have lost the unique plasmid present in the parent strain. In others this plasmid has suffered a deletion of a specific region of about 27 Md.

Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers. The patterns were compared with the patterns generated from strains of closely related species belonging to the "Bacillus cereus group" of bacteria, including B. cereus, Bacillus thuringiensis, and Bacillus mycoides. All B. anthracis profiles were identical for each of 18 restriction enzymes, each of 10 arbitrary PCR primers, and a repetitive extragenic palindrome-specific PCR primer. The PCR profiles generated with a coliphage M13-based primer exhibited slight pattern variation in a 400-to 500-bp band region. The B. anthracis profiles were unique compared with the profiles of the other species examined. In these other species, strain-to-strain variations were observed. Our results showed that isolates of B. anthracis are almost completely homogeneous, indicating a clonal lineage, and are distinct from other members of the B. cereus group and that B. anthracis, as a species in its own right, may have evolved only relatively recently.

SUMMARYHuman cytomegalovirus (HCMV) DNA was digested with restriction endonucleases and the fragments characterized with respect to molecular weight and relative mole proportions. The terminal fragments were identified by digesting HCMV DNA with exonucleases before restriction endonuclease treatment and subsequent gel analysis. The HindlII fragments of HCMV DNA were cloned in Escherichia coli and recombinant plasmids were characterized by digestion with restriction endonucleases and by molecular hybridization with HindlII, BgllI and XbaI fragments of the virus genome. Data from these experiments were used to construct physical maps of HCMV DNA for the HindlII, BgllI and XbaI restriction endonucleases. The terminal regions of the genome and the region containing fragment HindIII M were shown to be heterogeneous.