Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spollM gene

Smith, K. M.; Youngman, Philip

doi:10.1016/0300-9084(92)90143-3

Cited by 243 publications

(179 citation statements)

References 30 publications

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“…The LD 50 following intragastric inoculation of mice is unknown for either organism, but probably exceeds 10 10 organisms. Recombinant Lmdd-gag was constructed by stable modification of the bacterial chromosome using the shuttle vector pKSV7 [37] and a protocol modified from Camilli et al [38], as described [32], to insert HIV-gag into the sepA gene of Lmdd.…”

Section: Lmdd-gagmentioning

confidence: 99%

Live attenuated Listeria monocytogenes expressing HIV Gag: Immunogenicity in rhesus monkeys

Jiang

Rasmussen

Nolan

et al. 2007

Vaccine

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Induction of strong cellular immunity will be important for AIDS vaccine candidates. Natural infection with wild-type Listeria monocytogenes (Lm), an orally transmitted organism, is known to generate strong cellular immunity, thus raising the possibility that live attenuated Lm could serve as a vaccine vector. We sought to examine the potential of live attenuated Lm to induce cellular immune responses to HIV Gag. Rhesus macaques were immunized with Lmdd-gag that expresses HIV gag and lacks two genes in the D-alanine (D-ala) synthesis pathway. Without this key component of the bacterial cell wall, vaccine vector replication critically depends on exogenous D-ala. Lmdd-gag was given to animals either solely orally or by oral priming followed by intramuscular (i.m.) boosting; D-ala was co-administered with all vaccinations. Lmdd-gag and D-ala were well tolerated. Oral priming/oral boosting induced Gag-specific cellular immune responses, whereas oral priming/i.m. boosting induced systemic as well as mucosal anti-Gag antibodies. These results suggest that the route of vaccination may bias anti-Gag immune responses either towards T-helper type 1 (Th1) or Th2 responses; overall, our data show that live attenuated, recombinant Lmdd-gag was safe and immunogenic in primates.

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Section: Lmdd-gagmentioning

confidence: 99%

Live attenuated Listeria monocytogenes expressing HIV Gag: Immunogenicity in rhesus monkeys

Jiang

Rasmussen

Nolan

et al. 2007

Vaccine

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“…Such experiments are difficult to interpret, however, because it is not possible to prepare forespore fractions completely free of contamination from mother cell material (e.g., experiments reported in Table 4 of this work). The proposal that aE is activated exclusively in the mother cell compartment was motivated in part by the accumulation of genetic evidence that the protein roducts of genes now recognized to be members of the V regulon are required exclusively in the mother cell compartment (12,13,26,56,62,71,72). Direct evidence of compartmentspecific expression of a gene transcribed by EaE is most convincing with spoIlD, for which immunoelectron microscopy with colloidal gold-tagged antibody to P-galactosidase was used to demonstrate that expression of a lacZ fusion by a uE-controlled promoter was restricted to the mother cell compartment (14).…”

mentioning

confidence: 99%

“…Isolation of plasmid DNA from E. coli was carried out by the alkaline lysis procedure of Birnboim and Doly (5 (71). Spores were selected by incubation at 85°C for 20 min.…”

mentioning

confidence: 99%

“…If so, and if spolIM is expressed exclusively by &--associated RNA polymerase, then expression of spofIM in the forespore compartment should not be required for sporulation. To test this, we employed the plasmid integration-excision method of Illing et al (26), but with a temperature-sensitive integrational vector, pKSV7, as described previously (71). A 278-bp PvuII fragment from pKE35 internal to the spolIM coding sequence was inserted into pKSV7 to produce pKE47 (71).…”

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confidence: 99%

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Evidence that the spoIIM gene of Bacillus subtilis is transcribed by RNA polymerase associated with sigma E

Smith

Youngman

1993

J Bacteriol

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We have investigated the temporal and spatial regulation ofspoHIM, a gene ofBacillus subtilis whose product is required for complete septum migration and engulfment of the forespore compartment during sporulation. The spoHIM promoter was found to become active about 2 h after the initiation of sporulation. The effects of mutations on the expression of a spoIIM-lacZ fusion were most consistent with its utilization by &-Fassociated RNA polymerase (Eci). A unique 5' end of the in vivo spoHIM transcript was detected by primer extension analysis and was determined to initiate at the appropriate distance from a sequence conforming very closely to the consensus for genes transcribed by E&F. A partially purified preparation of E&X produced a transcript in vitro that initiated at the same nucleotide as the primer extension product generated from in vivo RNA. Ectopic induction of &E synthesis during growth resulted in the immediate and strong expression of a spoIIM-lacZ fusion, but an identical fusion was completely unresponsive to induced synthesis of either oF or rG under similar conditions. The results of plasmid integration-excision experiments in which the spolIM gene was reversibly disrupted by a temperature-sensitive integrational vector suggested that spolIM expression is required in the forespore compartment, but direct examination of subcellular fractions enriched for mother cell or forespore material indicated that spolIM expression cannot be confined to the forespore. We conclude that spoHIM is a member of the &E regulon and that it may be transcribed exclusively by Ecr-. We discuss the implications of this conclusion for models in which activation of o1r in the mother cell is proposed to be a part of the mechanism responsible for initiating separate programs of gene activity in the two sporangium compartments.Endospore formation in Bacillus subtilis involves a series of temporally and spatially ordered changes in cell morphology and gene expression (42,54). At a critical stage in this process, an asymmetric septation event creates two cell compartments, the mother cell and the forespore, each containing a transcriptionally active chromosome. At some point very soon after asymmetric septation, the mother cell and the forespore compartments begin to execute quite distinct programs of gene expression. The mechanisms responsible for establishing these compartment-specific programs are not fully understood, but they apparently involve, in part, the compartment-specific activation of RNA polymerase sigma factors (16,40,41,76). This is most obvious at the later stages of sporulation, in which a polymerase species associated with g is active exclusively in the mother cell compartment (11,36,43) and a species associated with cyG is active exclusively in the forespore (31, 78). At earlier stages, the situation is less certain and the basis for compartmentspecific gene activity is more speculative. Soon after asymmetric septation, a polymerase species associated with &rF becomes active in the forespore (45, 67). The o-F...

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“…A deleted copy of this gene, in which the reading frame and the first six and last ten codons were retained, was created using the splicing-by-overlap extension (SOE) PCR and primers listed in table 1 as previously described (Horton et al, 1990). The EGDe !lmo0501 DNA fragment was cloned into the temperature sensitive pKSV7 plasmid (Smith and Youngman, 1992), which was subsequently used in replacement of the EGDe chromosomal copy of lmo0501 based on a series of homologous recombination events . PCR analysis and DNA sequencing confirmed the lmo0501 locus deleted mutant.…”

Section: Culture Media and Bacterial Strainsmentioning

confidence: 99%

The lmo0501 gene coding for a putative transcription activator protein in Listeria monocytogenes promotes growth under cold, osmotic and acid stress conditions

2011

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In L. monocytogenes EGDe, the lmo0501 gene locus encodes a protein similar to the mannitol transcription regulator (MltR) protein in B. subtilis and B. stearothermophilus. In this study we investigated its functional role in L. monocytogenes EGDe cells in view of growth under different stress conditions. Increased lmo0501 gene expression at mRNA level was detected in response to cold, osmotic and organic acid stress exposure. An EGDe Δlmo0501 mutant strain was diminished in growth compared to the wild type strain in minimal defined medium containing either glucose or fructose, as carbon sources. Furthermore the lmo0501 null mutant had retarded growth under cold (4°C), salt (NaCl) and organic acid stress conditions compared to the parental wild type strain. Our results confirm the role of the lmo0501 gene in view of adaptation of L. monocytogenes cells to stress conditions as well as a contribution to the efficient utilization of glucose and fructose as carbon sources.Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-60467 Originally published at: Michel, E. The lmo0501 gene coding for a putative transcription activator protein in Listeria monocytogenes promotes growth under cold, osmotic and acid stress conditions. 2011, University of Zurich, Vetsuisse Faculty.

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Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spollM gene

Cited by 243 publications

References 30 publications

Live attenuated Listeria monocytogenes expressing HIV Gag: Immunogenicity in rhesus monkeys

Live attenuated Listeria monocytogenes expressing HIV Gag: Immunogenicity in rhesus monkeys

Evidence that the spoIIM gene of Bacillus subtilis is transcribed by RNA polymerase associated with sigma E

The lmo0501 gene coding for a putative transcription activator protein in Listeria monocytogenes promotes growth under cold, osmotic and acid stress conditions

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