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PubMed2025

The NagY antiterminator in Enterococcus faecalis: a novel regulatory mechanism and its impact on cell metabolism.

The Enterococcus genus is the most controversial group of lactic acid bacteria. While some strains are used as probiotics, other species, including Enterococcus faecalis, are responsible for health-related pathologies. Under conditions of infection, the N-acetylglucosamine metabolism of E. faecalis undergoes significant changes in expression, even more important than those of virulence factors. This metabolism is mediated by the nagY-nagE operon, which is regulated by the transcriptional antiterminator NagY. In this report, we focus on the regulatory mechanism of NagY and its impact on bacterial metabolism. We showed that NagY requires the interaction with the RNase III to achieve optimal induction of its own expression by cleaving the 5' untranslated region of the nagY mRNA. The NagY regulon was identified and the central role of the antiterminator in the E. faecalis metabolism was demonstrated, highlighting its importance in the opportunistic nature of the bacterium. This study provides a valuable advance in the understanding of regulation and the importance of post-transcriptional actors in E. faecalis adaptation.IMPORTANCEAs a commensal, Enterococcus faecalis colonizes the gastrointestinal tract of 31 to 80% of the intestinal microbiota in adults and is considered ubiquitous, due to its strong environmental stress resistance capabilities. However, in immunocompromised patients, the poorly understood transition from commensal to opportunistic pathogen occurs, and many studies suggest that the metabolism plays a central role in this process. In this study, we focus on the regulator NagY, which is involved in the metabolism of N-acetylglucosamine, an important carbon source for bacterial pathogens in the human host. We characterized a novel regulatory mechanism involving the NagY antiterminator and the ribonuclease RNase III. In addition, we identified the target genes of the regulator, through which we were able to demonstrate that NagY has a strong impact on the metabolism of β-glucosides. Overall, this work highlights the importance of regulation of the bacterial metabolic adaptation in the host.

Enterococcus faecalis↗
PubMed1979

Specificity of the bacteriophage lambda N gene product (pN): nut sequences are necessary and sufficient for antitermination by pN.

We have cloned the nutR site together with the tR1 site of bacteriophage lambda in the E. coli galactose operon to examine whether the lambda promoter sequences PR and PL are involved in the recognition specificity of the lambda N gene product (pN). We first constructed a derivative of plasmid pBR322 in which the expression of the tetracycline genes (tet) is controlled by the gal promoter (Pgal). This new plasmid contains a unique Hind III site between Pgal and tet into which the nutR and tR1 sites were introduced. The order of the relevant genetic markers in this second plasmid is Pgal-nutR-tR1-tet. Cells transformed with this plasmid express tet only if pN is provided and if the plasmid contains an intact gal promoter. Our data suggest that transcription which originates at Pgal is modified by pN at nutR, enabling it to pass through tR1 into tet. We conclude that promoters do not play a specific role in pN recognition and that nut sequences are both necessary and sufficient for pN action.

Bacteriophage lambda↗
PubMed1979

The site controlling the specificity of N action is outside the promoter-operator region: a triple hybrid phage lambda N21 imm434nin5.

A short interval of homology between imm lambda, imm434 and imm21 DNAs was identified near the leftward promoter-operator region. This homology, denoted Hs, was revealed by electron microscopic examination of lambda imm lambda/lambda imm21 and lambda imm434/lambda imm21 heteroduplexes, and permitted us to construct a special lambda hybrid (lambda hyB) which contains the N region of phage 21 and the adjacent imm region from phage 434. This triple hybrid, labmda N21 imm434nin5, was analysed by genetic, transcriptional and electronic micrographic techniques. Its leftward and rightward promoter-operator regions are of phage 434 specificity and are controlled by the 434 repressor. Surprisingly, the N21 gene of lambda hyB was found to be defective, perhaps to preserve the viability of the hybrid. Its leftward N-recognition system (nutL) is of phage 21 specificity since it responds only to the N21 function in complementation tests, as measured by antitermination of leftward transcription initiated at the pL promotor in the imm434 region. We conclude, therefore, that the pLoL region of 434 contains no information for the specificity of N antitermination. Both lambda imm21 and lambda hyB were found to be missing the tL1 terminator function (see also Salstrom and Szybalski, 1978b). In these phages, the tL2 terminator was found to be only 60% effective under N21 conditions, and therefore expression of their red-gam genes is sufficient to endow the lambda hyB and lambda N21- imm21nin5 phages with the Fec+ phenotype.

Coliphages↗
PubMed2026

Experimental evolution reveals genetic routes for adaptive loss of the antibacterial type VI secretion system.

The type VI secretion system (T6SS) is a contractile nanomachine used by Gram-negative bacteria to deliver effector proteins into target cells, contributing to both interbacterial competition and pathogenesis. Although T6SS gene clusters are present in recently isolated commensal and pathogenic Escherichia coli strains, they are absent from classical laboratory strains that have been propagated for decades in pure cultures, suggesting that T6SS can be lost in the absence of competition. Here, we combined experimental evolution with whole-genome sequencing to track the fate of the enteroaggregative Escherichia coli (EAEC) Sci1 T6SS during competition with either T6SS-susceptible or T6SS-immune bacteria. After ∼640 generations, T6SS activity was largely maintained during competition with T6SS-susceptible bacteria, whereas ∼90% of clones evolved with T6SS-immune bacteria lost or attenuated T6SS activity through diverse mutations within the sci1 promoter, essential T6SS structural genes, or the rfaH transcriptional antiterminator. We identified two RfaH-binding ops elements within the sci1 cluster, revealing antitermination as a regulatory element of EAEC T6SS transcription, which is conserved among Enterobacteriaceae. Our findings highlight how experimental evolution can reveal the selective forces shaping T6SS maintenance and identify new regulatory components controlling its activity.

Journal Article↗
PubMed1978

Control of lambda repressor prophage and establishment transcription by the product of gene tof.

Control of expression of the bacteriophage lambda (lambda) repressor was studied by measuring repressor transcription in noninduced and derepressed lambda lysogens. Three distinct modes of leftward transcription were observed from cI and the adjacent genes associated with the control of repressor synthesis: The prophage or maintenance mode Prm-cI-rex-ti repressor transcript occurs from repressed lysogens; the oop (Po-oop-to) transcript, and the lit (lit-ti) RNA, from the distal half of gene rex, both occur from induced tof+ prophage; the repressor establishment mode of transcription is observed throughout the rex-cI-tof-y-cII-oop interval between Po and ti from induced tof- prophage. The overall level of establishment mRNA synthesis is partially template dependent. However, the actual initiation step for repressor establishment transcription requires the participation of the lambda cIII, cII products, and also either requires the activity of Escherichia coli replication proteins, or is triggered by a replication initiation event. The cII cIII products do not positively stimulate de novo initiation of establishment transcription, but rather act after an initial replication-dependent step. Initiation of the establishment mode of repressor transcription is totally inhibited by more than 125-fold, in an all or none fashion, by the lambda antirepressor (Tof), the product of gene tof (cro). Since Tof only reduces the in vivo rightward transcription of cII from Pr by about 2-fold, we suggest that Tof inhibits repressor establishment transcription by either uncoupling the replication and cII-cIII dependent events, or by inhibiting the activity rather than the expression of the cIII, cII products. Our results do not fully support either of the present hypotheses that establishment transcription is initiated from the hypothetical Pre promoter in the y-interval, or arises through antitermination of the oop RNA. Since the initiation and control of the establishment mode of repressor transcription parallels the control of lit RNA synthesis, we propose a common mechanism underlies the initiation of these transcripts.

Coliphages↗
PubMed1977

Insertion sequence IS2 associated with int-constitutive mutants of bacteriophage lambda.

We have examined mutations in bacteriophage lambda called int-c, which confer elevated constitutive expression on the int gene for prophage integration. One class of mutations, which map between the b538 and bio386 endpoints, does not appear to be associated with any major chromosomal modification, whereas the second class has the IS2 insertion sequence in orientation II within the region between gene int and the b538 endpoint, All int-c mutations are within gene xis, with the possible exception of int-c548, which might be located between int and xis. The present data are most consistent with the following notion: (1) the point mutations of class one inactivate the tI terminator signal of the pI-tI leader RNA for gene int and thus render int expression independent of the antiterminating action of the cII and cIII products, and (2) the second class of int-c mutants is constitutive for Int because the IS2 insertion, when strategically located between int and tI, provides a new constitutive promoter for int transciption.

Coliphages↗
PubMed1979

Location of the regulatory site for establishment of repression by bacteriophage lambda.

During the lysogenic response to infection by bacteriophage lambda, the phage-specified cII and cIII proteins provide for the coordinate establishment of repression and integration of the viral DNA. One critical regulatory function of cII/cIII is an activation of synthesis of the cI protein, the repressor that maintains lysogeny. The mechanism and site for regulation of the cI gene by cII/cIII have been a subject of controversy. The two principal hypotheses for cII/cIII action are: initiation of new RNA chains in the y region of lambda DNA just to the left of the cII gene; or antitermination of a short leader RNA (4S or oop RNA) initiated to the right of the cII gene. In an effort to distinguish between these hypotheses, we have studied the cII-mediated turn-on of cI protein synthesis in three classes of prophage deletion strains: deletions of the 4S RNA promoter but not the y region, deletions that remove both regions, and deletions that leave both intact. We find that an intact y region is required for normal regulation of the cI gene by cII, but the 4S RNA promoter is not. From experiments with other mutants, we conclude that rightward transcription from the early lytic promoter is also not necessary for positive regulation. Our results suggest that positive regulation by cII/cIII involves initiation of new RNA chains through activation of promoter sites.

Binding Sites↗
PubMed1979

Sigma subunit of Escherichia coli RNA polymerase affects the function of lambda N gene.

A new class of Escherichia coli mutants, referred to as grn, has been isolated by localized mutagenesis. These mutations affect the sigma subunit of DNA-dependent RNA polymerase (ribonucleoside 5'-triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) by abolishing the expression of the lambda N gene, and they are closely lniked to dnaG in the order dnaG-grn-uxaA. Detailed study of one such mutant, grn1, yielded the following results: (i) grn1 is a single mutation and the mutant cell shows cold-sensitivity in growth; (ii) the Grn phenotype of the mutant can easily be suppressed by secondary mutations in the beta subunit gene of RNA polymerase; (iii) purified holoenzyme of RNA polymerase isolated from the mutant showed an altered salt-dependency in vitro, and the mixed reconstitution of the mutant with the wild-type subunits showed that the sigma subunit of the grn1 mutant is altered; (iv) lambda phage mutants (lambda grg), which overcome the grn mutation, can be classified into two groups, the "nin-deletion" and the "N-mutant" groups (both of these are also able to grow on the previously described groN mutant of Georgopoulos and nusAB of Friedman); (iv) the mutant polymerase transcribed 12S as well as 7S RNA from lambda DNA in the presence of the rho factor in vitro. These results indicate that the grn mutation alters the sigma subunit of RNA polymerase and that the sigma subunit participates in activating the N-mediated antitermination mode of lambda phage transcription.

Bacteriophage lambda↗
PubMed2023

Beyond antibiotic resistance: the whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria.

Pathogenic mycobacteria are a significant cause of morbidity and mortality worldwide. These bacteria are highly intrinsically drug resistant, making infections challenging to treat. The conserved whiB7 stress response is a key contributor to mycobacterial intrinsic drug resistance. Although we have a comprehensive structural and biochemical understanding of WhiB7, the complex set of signals that activate whiB7 expression remain less clear. It is believed that whiB7 expression is triggered by translational stalling in an upstream open reading frame (uORF) within the whiB7 5' leader, leading to antitermination and transcription into the downstream whiB7 ORF. To define the signals that activate whiB7, we employed a genome-wide CRISPRi epistasis screen and identified a diverse set of 150 mycobacterial genes whose inhibition results in constitutive whiB7 activation. Many of these genes encode amino acid biosynthetic enzymes, tRNAs, and tRNA synthetases, consistent with the proposed mechanism for whiB7 activation by translational stalling in the uORF. We show that the ability of the whiB7 5' regulatory region to sense amino acid starvation is determined by the coding sequence of the uORF. The uORF shows considerable sequence variation among different mycobacterial species, but it is universally and specifically enriched for alanine. Providing a potential rationalization for this enrichment, we find that while deprivation of many amino acids can activate whiB7 expression, whiB7 specifically coordinates an adaptive response to alanine starvation by engaging in a feedback loop with the alanine biosynthetic enzyme, aspC. Our results provide a holistic understanding of the biological pathways that influence whiB7 activation and reveal an extended role for the whiB7 pathway in mycobacterial physiology, beyond its canonical function in antibiotic resistance. These results have important implications for the design of combination drug treatments to avoid whiB7 activation, as well as help explain the conservation of this stress response across a wide range of pathogenic and environmental mycobacteria.

Preprint↗