Biology subjects

Verplaetse, E.

Publications and source records attributed to Verplaetse, E..

3 recordsLinked to original sources

bioRxiv2025

Paradigm shift for cry gene expression in Bacillus thuringiensis

In most Bacillus thuringiensis strains, the insecticidal cry genes are transcribed during sporulation by RNA-polymerases containing sigma factors E or K, leading to the formation of an insecticidal crystal within the mother cell along spore development. Some strains, such as the kurstaki HD1, a parent of commercial strains, also release insecticidal proteins Cry1I and Vip3A in the extracellular medium. vip3A gene expression is activated by the transcriptional regulator VipR at the onset of the stationary phase. Here, we expanded the VipR regulon to all the insecticidal-encoding genes of strain HD1 by identifying the VipR-binding box and conducting transcription assays. Unexpectedly, a VipR box was located in the promoter region of a putative N-acetylmuramoyl-L-alanine amidase gene upstream cry1Ac in strain kurstaki HD73, closely related to the HD1 but devoid of vipR. We demonstrated that introducing a functional vipR in this strain leads to the expression of the amidase-cry1Ac operon, resulting in an early and increased production of Cry1Ac. Investigating this phenotype, we showed that Cry1Ac was produced in a VipR-dependent manner in an HD73 {Delta}spo0A mutant. Similarly, an HD1{Delta}spo0A strain forms insecticidal crystals and produces all the insecticidal proteins encoded in its genome, including cry2Ab previously considered unexpressed in strain HD1. Finally, a genomic analysis revealed the presence of putative VipR-binding sequences upstream from insecticidal protein-encoding genes in several biopesticidal strains. Overall, our results break the dogma on the regulation of cry1A and cry2A genes and provide evidence of sporulation-independent Cry toxin production in biopesticidal Bt strains. ImportanceBacillus thuringiensis is a remarkably efficient entomopathogen due to its ability to produce various insecticidal proteins, such as Cry or Vip. This property has made it a highly effective biopesticide used worldwide. Our work breaks the dogma of cry1 and cry2 genes being regulated solely by sporulation-specific sigma factors and thus exclusively expressed during this process. Indeed, we demonstrated that the VipR regulator controls the transcription of all the insecticidal protein genes encoded by a strain closely related to that of commercial biopesticides and turns on their expression at the beginning of the stationary phase, leading to the production of insecticidal crystals independently of sporulation. By providing new knowledge on the regulation of insecticidal protein genes, these findings bring new insight for the genetic improvement of Bt strains used as commercial biopesticides.

microbiology↗

bioRxiv2022

Expression of the Bacillus thuringiensis vip3A insecticidal toxin gene is activated at the onset of stationary phase by VipR, an autoregulated transcription factor

The Vegetative insecticidal protein Vip3A is produced by some Bacillus thuringiensis strains from the mid-log growth phase to sporulation. Although Vip3A is important for the entomopathogenicity of B. thuringiensis, the vip3A gene regulation is unknown. In the B. thuringiensis kurstaki HD1 strain, vip3A is carried by the pBMB299 plasmid, which is absent in the closely related strain B. thuringiensis kurstaki HD73. Using a transcriptional fusion between the vip3A promoter and lacZ, we observed that the HD73 strain is unable to express vip3A. This result suggests that a specific regulator is required for vip3A expression. Assuming that the regulator gene is located on the same plasmid as vip3A, we transferred the pBMB299 from the HD1 strain to the HD73 strain. We found that Vip3A was produced in the HD73 strain containing pBMB299, suggesting that the regulator gene is located on this plasmid. Using this heterologous host and promoter-lacZ transcription fusions, we confirmed that VipR is essential to activate vip3A expression at the onset of stationary phase. We demonstrated that vipR transcription is positively autoregulated and the determination of the vipR and vip3A promoters pinpointed a putative VipR target upstream from the Sigma A-specific -10 region of these two promoters. Surprisingly, this conserved sequence was also found upstream of cry1I and cry2 genes. Finally, we showed that vip3A and vipR expression is drastically increased in a {Delta}spo0A mutant unable to initiate sporulation. In conclusion, we have characterized a novel regulator involved in the entomopathogenic potency of B. thuringiensis through a sporulation-independent pathway. ImportanceThe insecticidal properties of Bacillus thuringiensis are mainly due to Cry toxins which form a crystalline inclusion during sporulation. However, other proteins participate in the pathogenicity of the bacterium, notably the Vip3A toxins that are produced from vegetative growth to sporulation. The VipR regulator that activates vip3A gene expression at the onset of stationary phase is positively autoregulated and analysis of the promoter region of the vip3A and vipR genes reveals the presence of a highly conserved DNA sequence. This possible VipR target sequence is also found upstream of the cry2A and cry1I genes, suggesting that Cry toxins can be produced before the bacteria enter sporulation. Such a result could allow us to better understand the role of Cry and Vip3A toxins during the B. thuringiensis infectious cycle in insects, in addition to the primary role of the Cry toxins in the toxemia caused by ingestion of crystals.

microbiology↗

bioRxiv2019

Heme uptake in Lactobacillus sakei evidenced by a new ECF-like transport system.

Lactobacillus sakei is a non-pathogenic lactic acid bacterium and a natural inhabitant of meat ecosystems. Although red meat is a heme-rich environment, L. sakei does not need iron or heme for growth, while possessing a heme-dependent catalase. Iron incorporation into L. sakei from myoglobin and hemoglobin was formerly shown by microscopy and the L. sakei genome reveals a complete equipment for iron and heme transport. Here, we report the characterization of a five-gene cluster (lsa1836-1840) encoding a putative metal iron ABC transporter. Interestingly, this cluster, together with a heme dependent catalase gene, is also conserved in other species from the meat ecosystem. Our bioinformatic analyses revealed that the locus might refer to a complete machinery of an Energy Coupling Factor (ECF) transport system. We quantified in vitro the intracellular heme in wild-type (WT) and in our {Delta}lsa1836-1840 deletion mutant using an intracellular heme sensor and ICP-Mass spectrometry for quantifying incorporated 57Fe heme. We showed that in the WT L. sakei, heme accumulation occurs fast and massively in the presence of hemin, while the deletion mutant was impaired in heme uptake; this ability was restored by in trans complementation. Our results establish the main role of the L. sakei Lsa1836-1840 ECF-like system in heme uptake. This research outcome shed new light on other possible functions of ECF-like systems. ImportanceLactobacillus sakei is a non-pathogenic bacterial species exhibiting high fitness in heme rich environments such as meat products, although it does not need iron nor heme for growth. Heme capture and utilization capacities are often associated with pathogenic species and are considered as virulence-associated factors in the infected hosts. For these reasons, iron acquisition systems have been deeply studied in such species, while for non-pathogenic bacteria the information is scarce. Genomic data revealed that several putative iron transporters are present in the genome of the lactic acid bacterium L. sakei. In this study, we demonstrate that one of them, is an ECF-like ABC transporter with a functional role in heme transport. Such evidence has not yet been brought for an ECF, therefore our study reveals a new class of heme transport system.

microbiology↗