Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes.

Menaquinone (MK) is a non-protein component of the Bacillus subtilis (Bs) electron transport chain synthesized from chorismate through a series of MK-specific reactions. The genes encoding biosynthesis of the naphthoquinone ring of MK are clustered at 273 degrees on the Bs chromosome. A 3.9-kb region capable of rescuing men mutants blocked in the early stages of MK biosynthesis was sequenced and found to contain three major open reading frames (ORFs). The first ORF (menF) has a predicted size of 51.8 kDa and 34% amino-acid identity with the isochorismate synthases of Escherichia coli (EntC) and Aeromonas hydrophila (AmoA), ORF2 (menD) a predicted size of 60.2 kDa and 21% identity with MenD of E. coli. ORF3 has a predicted size of 21.4 kDa and 29% identity to triacylglycerol lipase of Psychrobacter immobilis. No sequence corresponding to menC was identified. Plasmid integrational studies of the men gene cluster had suggested the presence of promoters secondary to the previously identified p1 men promoter. Sequence analysis revealed a putative promoter region upstream from ORF3.

Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes.

Under iron-limiting conditions, Bacillus subtilis (Bs) produces the siderophore 2,3-dihydroxybenzoate (DHB) to acquire extracellular iron. In Escherichia coli (Ec), DHB is a precursor of the siderophore enterobactin, which suggested that Bs may possess similar biosynthetic enzymes. The sequences of two overlapping Bs clones capable of complementing Ec enterobactin mutants [Grossman, T.H., Tuckman, M., Ellestad, S. and Osburne, M.S. (1993) Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: Relationship between B. subtilis sfpo and Escherichia coli entD genes. J. Bacteriol. 175, 6203-6211] were analyzed and five open reading frames were identified. These genes are located near 291 degrees on the Bs chromosome and have been termed dhbA, dhbC, dhbE, dhbB and dhbF, based on similarities to Ec ent homologs. Amino-acid identities between gene product homologs are: EntA and DhbA, 41%; EntC and DhbC, 35%; EntE and DhbE, 48%; EntB and DhbB, 54%; and EntF and DhbF, 29%. DhbC is also 35% identical to the Bs menaquinone-specific isochorismate synthase, MenF, illustrating an example of gene duplication. Operon disruption studies suggested that the dhb genes comprise an operon of at least four genes.

Drug resistance in tuberculosis.

Drug-resistant tuberculosis remains a worldwide problem. New laboratory methods have improved our ability to more rapidly identify resistant strains, but the most effective approach is to prevent the appearance of resistance by appropriate choice of antibiotics and directly-observed therapy. Mycobacterium tuberculosis is treated with familiar and unique drugs; consequently, mechanisms of resistance have some unique features. All drug resistance thus far identified develops by mutational events rather than acquisition of resistance genes from other bacteria. An agenda is presented for countering the appearance of further drug resistance in mycobacteria.

Analysis of Mycobacterium tuberculosis transmission patterns in a homeless shelter outbreak.

SETTING: From July 1997 through May 1998, ten tuberculosis (TB) cases were reported among men in a Syracuse New York homeless shelter for men. OBJECTIVE AND DESIGN: Investigation to determine extent of, and prevent further, transmission of Mycobacterium tuberculosis. RESULTS: Epidemiologic and laboratory evidence suggests that eight of the ten cases were related. Seven cases had isolates with matching six-band IS6110 DNA fingerprints; the isolate from another case had a closely related fingerprint pattern and this case was considered to be caused by a variant of the same strain. Isolates from eight cases had identical spoligotypes. The source case had extensive cavitary disease and stayed at the shelter nightly, while symptomatic, for almost 8 months before diagnosis. A contact investigation was conducted among 257 shelter users and staff, 70% of whom had a positive tuberculin skin test, including 21 with documented skin test conversions. CONCLUSIONS: An outbreak of related TB cases in a high-risk setting was confirmed through the use of IS6110 DNA fingerprinting in conjunction with spoligotyping and epidemiologic evidence. Because of the high rate of infection in the homeless population, routine screening for TB and preventive therapy for eligible persons should be considered in shelters.

Sporulation properties of cytochrome a-deficient mutants of Bacillus subtilis.

Three classes of cytochrome a-deficient mutants of Bacillus subtilis have been found to be asporogenic or oligosporogenic. All three classes showed declines in adenosine 5'-triphosphate (ATP) concentrations during early sporulation, at a time when ATP levels in wild-type strains are constant. Class III mutants were found to be deficient in aconitase and isocitric dehydrogenase, and showed reduced maximum growth in nutrient sporulation medium. These mutants also suffered the most rapid decline in ATP concentration in early sporulation, and exhibited neither the biphasic oxygen consumption curve nor the increase in pH normally observed at the end of logarithmic growth in nutrient sporulation medium. Nicotinamide adenine dinucleotide oxidase activities of purified membrane preparations were approximately normal for mutants in all classes, except for two of the class II mutants and one class III mutant. Neither cytochrome a nor cytochrome c appears to be an obligatory intermediate in cyanide-sensitive nicotinamide adenine dinucleotide oxidation in B. subtilis.

Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis.

Cytochrome aa3 is one of two terminal oxidase complexes in the Bacillus subtilis electron transport chain. A novel genetic strategy was devised which permitted the isolation of B. subtilis mutants lacking cytochrome aa3 by selection for streptomycin-resistant clones which failed to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Two mutations were studied intensively. Spectroscopic examination showed that each mutant lacked cytochrome aa3; they were also asporogenous and unable to grow on lactate as the sole carbon and energy source. These mutations were mapped to a locus designated ctaA, located at 127 degrees between pyrD and metC on the B. subtilis chromosome. Both ctaA mutations were closely linked by transformation to the pycA locus. The ctaA locus and a portion of the pycA locus were cloned from a B. subtilis integration library constructed in Escherichia coli. A recombinant plasmid containing a 4.0-kilobase insert of B. subtilis DNA could transform both ctaA mutants to CtaA+. Gene disruption and complementation experiments with subcloned fragments revealed that the ctaA locus consisted of a single transcriptional unit about 1.35 kilobase pairs in size. The nucleotide sequence of the ctaA transcriptional unit contains a single open reading frame capable of coding for a protein with a predicted molecular weight of 34,065. The predicted protein is extremely hydrophobic, with several probable membrane-spanning domains. No sequence similiarity was found between ctaA and the highly conserved procaryotic and mitochondrial oxidase polypeptides. Cloning and sequence analysis of two ctaA mutations revealed that one allele is a nonsense mutation in the carboxy terminus and the other is a missense mutation in the amino terminus; this indicates that the pleiotropic phenotype conferred by each mutation was caused by loss of CtaA or of its activity. Genetic evidence suggests that the ctaA gene product is required as an accessory protein in the genetic expression, posttranslational biogenesis, or both, of the cytochrome aa3 complex and during an early stage of sporogenesis.

Structure and expression of the cytochrome aa3 regulatory gene ctaA of Bacillus subtilis.

Mutations that define the ctaA gene of Bacillus subtilis block cytochrome aa3 formation and sporulation. We have recently described the isolation and initial characterization of the ctaA locus. Analysis of in vivo mRNA transcripts by RNase protection experiments located the 5' and 3' termini of the ctaA transcript, confirming a monocistronic structure. By using a nuclease protection assay, an increase in the abundance of steady-state ctaA mRNA was observed during the initiation of sporulation, followed by a decrease during subsequent stages. Transcripts originating from the ctaA gene were most abundant 2.0 h after the end of exponential growth. This pattern of ctaA mRNA accumulation was confirmed by coupling the transcription of the ctaA gene to lacZ in an integrative plasmid vector. Expression of ctaA was not repressed by glucose and was independent of the spoOA and spoOH (sigH) gene products. Postexponential expression was found to be dependent on the product of the strC gene. The expression of ctaA appears to be regulated in a growth stage-specific manner. The transcriptional start site, identified by high-resolution S1 nuclease protection experiments, was preceded by a single sigma A-dependent promoter sequence.

Correlation between cytochrome aa3 concentrations and streptomycin accumulation in Bacillus subtilis.

Accumulation of aminoglycosides by Bacillus subtilis appears to require specific components of the electron transport chain. These components include cytochromes and the lipophilic quinone vitamin K2. The present study concerns the importance of cytochrome aa3, a terminal oxidase, in the uptake of streptomycin. Growth conditions have been established such that the concentration of cytochrome aa3 can be modified over a wide range; on defined minimal salts agar, the wild-type strain (RB1) and an strC mutant (RB95) synthesized cytochrome aa3 only when adequate amounts of Casamino Acids (Difco Laboratories, Detroit, Mich.) were present. A positive correlation between cytochrome aa3 levels and streptomycin accumulation was observed. The same correlation was seen when cytochrome aa3 was measured in relation to growth susceptibility. These correlations suggest that cytochrome aa3 is necessary for accumulation of streptomycin by B. subtilis.

Isolation of F' plasmids carrying a portion of the Salmonella typhimurium histidine transport operon.

The transposable drug resistance element Tn10 was employed as a region of homology to direct the insertion of Tn10-containing derivatives of F'ts114 lac into the chromosome of a Salmonella typhimurium strain that carries a Tn10 insertion in the histidine transport operon. Based on the direction of transfer of the resulting Hfr strains, the chromosomal Tn10 insertion was determined to be in orientation "A." New F' plasmids were selectively generated from one of the Hfr strains. The F' factors carry an intact dhuA hisJ portion of the histidine transport operon. A Southern hybridization revealed that one of the F' plasmids was formed by a type II excision event.

Streptomycin accumulation by Bacillus subtilis requires both a membrane potential and cytochrome aa3.

Cytochrome aa3 concentrations in the cytoplasmic membrane of Bacillus subtilis were altered by growth conditions, and the effects on the membrane potential (delta psi) in whole cells were measured. When cytochrome aa3 was absent, the magnitude of delta psi was not diminished by comparison with the delta psi measured in cells containing normal cytochrome aa3 concentrations. In addition, the energy-dependent uptake of proline and glutamate was comparable at both cytochrome aa3 concentrations. However, in the cytochrome aa3-deficient cell preparation, accumulation of the aminoglycoside antibiotic streptomycin was much lower than that of the cytochrome aa3-sufficient cells. When cells were cultured under conditions that stimulated higher than normal concentrations of cytochrome aa3, delta psi was also increased, and enhanced streptomycin accumulation was observed. Phenazine methosulfate-ascorbate was used both in delta psi measurements and in uptake studies to provide high rates of electron transport and maximal delta psi values. These results, taken together with those previously published (A. S. McEnroe and H. W. Taber, Antimicrob. Agents Chemother. 26:507-512, 1984) suggest that the uptake of streptomycin by B. subtilis requires adequate levels both of delta psi and cytochrome aa3.

Multiple-aminoglycoside-resistant mutants of Bacillus subtilis deficient in accumulation of kanamycin.

Three classes of spontaneous multiple-aminoglycoside-resistant (mar) mutants of Bacillus subtilis were isolated by plating on a low (1.2 mug/ml) concentration of kanamycin sulfate and were found to be resistant also to low concentrations of paromomycin, neomycin and gentamicin. The three classes could be distinguished one from another by their degree of cytochrome deficiency, respiration deficiency, and susceptibility to kanamycin lethality. A fluctuation test showed that the mutations were spontaneous and not induced by the conditions of selection. Representative strains from two classes of mutants (mar-2 and mar-3) accumulated aminoglycoside very poorly in comparison with the parent strain, whereas a strain of the third class (mar-1) inactivated aminoglycoside present in the growth medium. The mar-3 strain studied (aroD163) had previously been shown to be a menaquinone auxotroph (Farrand and Taber, 1973) and to be deficient in amino acid uptake (Bisschop et al., 1975). Such mutants, which are resistant to low concentrations of aminoglycosides, may be of use in elucidating the biochemical and genetic bases of certain bacterial transport systems.

Transcriptional regulation of the Bacillus subtilis menp1 promoter.

The Bacillus subtilis men genes encode biosynthetic enzymes for formation of the respiratory chain component menaquinone. The menp1 promoter previously was shown to be the primary cis element for menFD gene expression. In the present work, it was found that either supplementation with nonfermentable carbon sources or reutilization of glycolytic end products increased menp1 activity in the late postexponential phase. The effect on menp1 activity by a particular end product (such as acetoin or acetate) was prevented by blocking the corresponding pathway for end product utilization. Alteration of a TGAAA motif within the promoter region resulted in unregulated menp1 activity throughout the culture cycle, irrespective of the carbon source added.

Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosyntheses of menaquinone and 2,3-dihydroxybenzoate.

Bacillus subtilis has duplicate isochorismate synthase genes, menF and dhbC. Isochorismate synthase is involved in the biosynthesis of both the respiratory chain component menaquinone (MK) and the siderophore 2,3-dihydroxybenzoate (DHB). Several menF and dhbC deletion mutants were constructed to identify the contribution made by each gene product to MK and DHB biosynthesis. menF deletion mutants were able to produce wild-type levels of MK and DHB, suggesting that the dhbC gene product is able to compensate for the lack of MenF. However, a dhbC deletion mutant produced wild-type levels of MK but was DHB deficient, indicating that MenF is unable to compensate for the lack of DhbC. A menF dhbC double-deletion mutant was both MK and DHB deficient. Transcription analysis showed that expression of dhbC, but not of menF, is regulated by iron concentration. A dhbA'::lacZ fusion strain was constructed to examine the effects of mutations to the iron box sequence within the dhb promoter region. These mutations abolished the iron-regulated transcription of the dhb genes, suggesting that a Fur-like repressor protein exists in B. subtilis.

Biosynthesis of o-succinylbenzoic acid in Bacillus subtilis: identification of menD mutants and evidence against the involvement of the alpha-ketoglutarate dehydrogenase complex.

The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacillus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) synthase-alpha-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed E1, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the alpha-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate alpha-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity.

Pleiotropic menaquinone-deficient mutant of Bacillus subtilis.

A multiple aromatic amino acid auxotroph of Bacillus subtilis 168 has been isolated which is unable to synthesize menaquinone-7 (MK-7) unless supplied with shikimic acid (SHK). The mutant, RB163, was isolated by selecting for resistance to low levels (1.5 mug/ml) of kanamycin. Enzymatic and genetic analyses show that the strain is an aroD mutant lacking 5-dehydroshikimate reductase. Under growth conditions in which its MK-7 deficiency is expressed, RB163 is deficient in cytochromes a, b, and c, exhibits low growth yields, and does not sporulate. Genetic analysis indicates that this pleiotropic phenotype is the result of a single genetic event. All phenotypic characteristics are reversible when the mutant is grown under conditions such that MK is synthesized. Comparison of strain RB163 with other aro mutants blocked before SHK ("early-aro" mutants) reveals interesting differences. Most early-aro mutants are cytochrome- and MK-sufficient, sporogenous, and sensitive to kanamycin when grown in the absence of SHK. However, in addition to strain RB163, two other aro mutants were found to show the pleiotropic phenotype. These three mutants have in common, and differ from other early-aro strains in, the inability to synthesize MK. It is suggested that the phenotypically wild-type aro mutants are bradytrophic, allowing enough substrate flow through the common aromatic pathway to satisfy the MK requirement. The pleiotropic mutants are thought to be completely blocked in the common pathway, thus accounting for their inability to synthesize MK.