Forespore-Specific Transcription of thelonB Gene during Sporulation in Bacillus subtilis

ABSTRACT The Bacillus subtilis genome encodes two members of the Lon family of prokaryotic ATP-dependent proteases. One, LonA, is produced in response to temperature, osmotic, and oxidative stress and has also been implicated in preventing ςG activity under nonsporulation conditions. The second is encoded by thelonB gene, which resides immediately upstream fromlonA. Here we report that transcription of lonBoccurs during sporulation under ςF control and thus is restricted to the prespore compartment of sporulating cells. First, expression of a lonB-lacZ transcriptional fusion was abolished in strains unable to produce ςF but remained unaffected upon disruption of the genes encoding the early and late mother cell regulators ςE and ςK or the late forespore regulator ςG. Second, the fluorescence of strains harboring a lonB-gfp fusion was confined to the prespore compartment and depended on ςF production. Last, primer extension analysis of the lonB transcript revealed −10 and −35 sequences resembling the consensus sequence recognized by ςF-containing RNA polymerase. We further show that thelonB message accumulated as a single monocistronic transcript during sporulation, synthesis of which required ςF activity. Disruption of the lonB gene did not confer any discernible sporulation phenotype to otherwise wild-type cells, nor did expression of lonB from a multicopy plasmid. In contrast, expression of a fusion of the lonB promoter to the lonA gene severely reduced expression of the ςG-dependent sspE gene and the frequency of sporulation. In confirmation of earlier observations, we found elevated levels of ςF-dependent activity in aspoIIIE47 mutant, in which the lonB region of the chromosome is not translocated into the prespore. Expression of either lonB or the P lonB -lonAfusion from a plasmid in the spoIIIE47 mutant reduced ςF -dependent activity to wild-type levels. The results suggest that both LonA and LonB can prevent abnormally high ςF activity but that only LonA can negatively regulate ςG.

Early Cephamycin Biosynthetic Genes Are Expressed from a Polycistronic Transcript in Streptomyces clavuligerus

ABSTRACT A polycistronic transcript that is initiated at the latpromoter has been implicated in the expression of the genes involved in early steps of cephamycin C biosynthesis in Streptomyces clavuligerus. pcbC is also expressed as a monocistronic transcript from its own promoter. However, an alternative interpretation involving expression via three separate yet interdependent transcripts has also been proposed. To distinguish between these possibilities, mutants lacking the latpromoter and containing a transcription terminator within thelat gene (Δlat::tsr/term mutants) were created. This mutation eliminated the production of lysine-ɛ-aminotransferase (the lat gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) (the pcbAB gene product) was eliminated in Δlat::tsr/term mutants, while production of isopenicillin N synthase (IPNS) (the pcbCgene product) was greatly reduced. The provision of α-aminoadipate to the Δlat::tsr/term mutants, either via exogenous feeding or via lat gene complementation, did not restore production of ACVS or IPNS. Analysis of RNA isolated from the Δlat::tsr/term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic pcbC transcript levels were greatly decreased. In contrast, Δlat mutants created by in-frame internal deletion of lat maintained the polycistronic transcript and allowed production of wild-type levels of both ACVS and IPNS.

glmM Operon and Methicillin-ResistantglmM Suppressor Mutants in Staphylococcus aureus

ABSTRACT The Staphylococcus aureus phosphoglucosamine mutase gene glmM was shown to be the last gene of a three-cistron operon, orf1-orf2-glmM. One transcriptional start was identified upstream of orf1, and a second start producing a monocistronic transcript was identified upstream of glmM. Disruption of glmM abolished GlmM production, decreased methicillin resistance, and resulted in teicoplanin hypersusceptibility without affecting the production of the endogenous penicillin-binding proteins and PBP 2′. Complementation of the glmM mutation by the complete glmMoperon restored both methicillin resistance and normal teicoplanin susceptibility. In contrast, a highly methicillin-resistant suppressor mutant obtained by selection for growth in the presence of methicillin remained GlmM deficient and teicoplanin hypersusceptible. The suppressor mutation was not linked to the glmM operon but was correlated with decreased autolysis and increased production of a 49-kDa protein, suggesting that there is an alternative pathway for glucosamine-1-phosphate synthesis in S. aureus.

The Nine Genes of the Nocardia lactamdurans Cephamycin Cluster Are Transcribed into Large mRNAs from Three Promoters, Two of Them Located in a Bidirectional Promoter Region

ABSTRACT The nine biosynthesis genes of the Nocardia lactamdurans cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, andpcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp andcefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC,cmcI, cmcJ, cefF, andcmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp.

Identification and Expression of the Bacillus subtilis Fructose-1,6-Bisphosphatase Gene (fbp)

ABSTRACT The Bacillus subtilis fbp gene encoding fructose-1,6-bisphosphatase (FBPase) was originally identified asyydE. The fbp gene was expressed at a fairly constant level in cells undergoing glycolysis or gluconeogenesis.fbp transcription was initiated 94 bp upstream of the translation initiation codon, resulting in a 2.4-kb monocistronic transcript. Interestingly, B. subtilis FBPase exhibited no significant similarity to other FBPases in protein sequence databases.

Clustered Genes Encoding the Methyltransferases of Methanogenesis from Monomethylamine

ABSTRACT Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA inMethanosarcina barkeri MS. The gene encoding MMCP (mtmC) is located directly upstream of the gene encoding MMAMT (mtmB). The gene encoding MT2-A (mtbA) was found 1.1 kb upstream of mtmC, but no obvious open reading frame was found in the intergenic region betweenmtbA and mtmC. A single monocistronic transcript was found for mtbA that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried mtmCB. The larger transcript also encoded mtmP, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of mtmC. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame ofmtmB was interrupted by a single in-frame, midframe, UAG codon which was also found in mtmB from M. barkeri NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression ofmtmB in this methanogen.

A monocistronic transcript for a trypanosome variant surface glycoprotein.

Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.

A monocistronic transcript for a trypanosome variant surface glycoprotein

Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.