Regulation of Leaderless mRNA Translation in Bacteria

Leiva, Lorenzo Eugenio; Katz, Assaf

doi:10.3390/microorganisms10040723

Cited by 16 publications

(8 citation statements)

References 63 publications

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“…Both codons are frequently used in C. glutamicum (43.8% and 56.2%, respectively) [ 34 ], so we cannot expect a major effect on ArgT translation due to altered codon usage here. As stated above, argT is a leaderless transcript and until now it is not fully understood how translation initiation and regulation works for these transcripts [ 35 ]. It is known, however, that downstream elements such as CA multimers can improve translation speed in E. coli [ 36 ], presumably through the provision of a lack of structure, since secondary mRNA structures immediately downstream of the AUG were shown to influence translation efficiency of leaderless mRNA [ 37 ].…”

Section: Resultsmentioning

confidence: 99%

Discovery of novel amino acid production traits by evolution of synthetic co-cultures

et al. 2023

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Background Amino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities. Results Here we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either l-leucine or l-arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The l-leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na+/H+-antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to l-leucine transport. The l-arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only l-arginine uptake system of C. glutamicum with an affinity of KD = 30 nM. Finally, deletion of argTUV in an l-arginine producer strain resulted in a faster and 24% higher l-arginine production in comparison to the parental strain. Conclusion Our work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.

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Section: Resultsmentioning

confidence: 99%

Discovery of novel amino acid production traits by evolution of synthetic co-cultures

et al. 2023

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“…Alternatively, TSS-1 may be an actual initiation site of transcription, and its close location to the ATG start codon may implicate the nos transcript as potentially having a leaderless organization with no identifiable SD sequence, a phenomenon not well characterized in S. aureus . Leaderless mRNAs permit translation without an identifiable SD sequence ( 47 ). It remains unclear exactly how translation is initiated from these leaderless transcripts, but several necessary elements have been implicated, including a local absence of secondary structure and simply the 5′ AUG of the mRNA ( 48 , 49 ).…”

Section: Resultsmentioning

confidence: 99%

Factors impacting the regulation of nos gene expression in Staphylococcus aureus

Brandwein,

Sculthorpe,

Ridder

et al. 2023

Microbiol Spectr

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Staphylococcus aureus nitric oxide synthase (saNOS) contributes to oxidative stress resistance, antibiotic tolerance, virulence, and modulation of aerobic and nitrate-based cellular respiration. Despite its involvement in these essential processes, the genetic regulation of nos expression has not been well characterized. 5′ rapid amplification of cDNA ends on nos RNA isolated from S. aureus UAMS-1 (USA200 strain) and AH1263 (USA300 strain) revealed that the nos transcriptional start site mapped to an adenine nucleotide in the predicted Shine-Dalgarno site located 11 bp upstream of the nos ATG start codon, suggesting that the nos transcript may have a leaderless organization or may be subject to processing. The SrrAB two-component system (TCS) was previously identified as a positive regulator of nos RNA levels, and experiments using a β-galactosidase reporter plasmid confirmed that SrrAB is a positive regulator of nos promoter activity. In addition, the quorum-sensing system Agr was identified as a negative regulator of low-oxygen nos expression in UAMS-1, with activity epistatic to SrrAB. Involvement of Agr was strain dependent, as nos expression remained unchanged in an AH1263 agr mutant, which has higher Agr activity compared to UAMS-1. Furthermore, nos promoter activity and RNA levels were significantly stronger in AH1263 relative to UAMS-1 during late-exponential low-oxygen growth, when nos expression is maximal. Global regulators Rex and MgrA were also implicated as negative regulators of low-oxygen nos promoter activity in UAMS-1. Collectively, these results provide new insight into factors that control nos expression. IMPORTANCE Bacterial nitric oxide synthase (bNOS) has recently emerged in several species as a key player in resistance to stresses commonly encountered during infection. Although Staphylococcus aureus (sa)NOS has been suggested to be a promising drug target in S. aureus , an obstacle to this in practice is the existence of mammalian NOS, whose oxygenase domain is like bacterial NOS. Increased understanding of the nos regulatory network in S. aureus could allow targeting of saNOS through its regulators, bypassing the issue of also inhibiting mammalian NOS. Furthermore, the observed strain-dependent differences in S. aureus nos regulation presented in this study reinforce the importance of studying bacterial NOS regulation and function at both the strain and species levels.

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“…Both cvkR and cas12k were found to be expressed from leaderless mRNAs. Compared to eukaryotes and archaea, the frequency of leaderless mRNAs in bacteria, especially in gram-negative bacteria, is rather low 53 – 55 . The dRNA-seq analysis of transcriptomes from 14 different growth conditions provided evidence for 51 leaderless mRNAs in the cyanobacterium Synechocystis 6803 56 .…”

Section: Discussionmentioning

confidence: 97%

CvkR is a MerR-type transcriptional repressor of class 2 type V-K CRISPR-associated transposase systems

et al. 2023

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Certain CRISPR-Cas elements integrate into Tn7-like transposons, forming CRISPR-associated transposon (CAST) systems. How the activity of these systems is controlled in situ has remained largely unknown. Here we characterize the MerR-type transcriptional regulator Alr3614 that is encoded by one of the CAST (AnCAST) system genes in the genome of cyanobacterium Anabaena sp. PCC 7120. We identify a number of Alr3614 homologs across cyanobacteria and suggest naming these regulators CvkR for Cas V-K repressors. Alr3614/CvkR is translated from leaderless mRNA and represses the AnCAST core modules cas12k and tnsB directly, and indirectly the abundance of the tracr-CRISPR RNA. We identify a widely conserved CvkR binding motif 5’-AnnACATnATGTnnT-3’. Crystal structure of CvkR at 1.6 Å resolution reveals that it comprises distinct dimerization and potential effector-binding domains and that it assembles into a homodimer, representing a discrete structural subfamily of MerR regulators. CvkR repressors are at the core of a widely conserved regulatory mechanism that controls type V-K CAST systems.

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Regulation of Leaderless mRNA Translation in Bacteria

Cited by 16 publications

References 63 publications

Discovery of novel amino acid production traits by evolution of synthetic co-cultures

Discovery of novel amino acid production traits by evolution of synthetic co-cultures

Factors impacting the regulation of nos gene expression in Staphylococcus aureus

CvkR is a MerR-type transcriptional repressor of class 2 type V-K CRISPR-associated transposase systems

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