Daniela Ghisotti

Transcriptional and post‐transcriptional control of polynucleotide phosphorylase during cold acclimation in Escherichia coli

Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is one of the cold shock‐induced proteins in Escherichia coli and pnp, the gene encoding it, is essential for growth at low temperatures. We have analysed the expression of pnp upon cold shock and found a dramatic transient variation of pnp transcription profile: within the first hour after temperature downshift the amount of pnp transcripts detectable by Northern blotting increased more than 10‐fold and new mRNA species that cover pnp and the downstream region, including the cold shock gene deaD, appeared; 2 h after temperature downshift the transcription profile reverted to a preshift‐like pattern in a PNPase‐independent manner. The higher amount of pnp transcripts appeared to be mainly due to an increased stability of the RNAs. The abundance of pnp transcripts was not paralleled by comparable variation of the protein: PNPase steadily increased about twofold during the first 3 h at low temperature, as determined both by Western blotting and enzymatic activity assay, suggesting that PNPase, unlike other known cold shock proteins, is not efficiently translated in the acclimation phase. In experiments aimed at assessing the role of PNPase in autogenous control during cold shock, we detected a Rho‐dependent termination site within pnp. In the cold acclimation phase, termination at this site depended upon the presence of PNPase, suggesting that during cold shock pnp is autogenously regulated at the level of transcription elongation.

Multicenter trial on mother-to-infant transmission of GBV-C virus

Evidence indicates that the GBV‐C or hepatitis G virus can cause persistent infection in humans, but little is known on the importance of vertical transmission. To assess the risk of mother‐to‐infant transmission and the clinical outcome of infected babies, we investigated 175 anti‐HCV positive mothers and followed‐up their children for 3–33 months. GBV‐C RNA was detected by RT‐PCR and anti‐E2 antibody was assayed by EIA. Thirty‐four (19.4%) women were GBV‐C RNA positive and transmission occurred to 21 (61.8%) babies; 20 (95.2%) acquired GBV‐C alone, and one (4.8%) GBV‐C and HCV. Maternal factors such as intravenous drug use, HIV coinfection, HCV‐RNA positivity, and type of feeding were not correlated with GBV‐C transmission. GBV‐C RNA remained persistently positive in all infected babies but one baby who seroconverted to anti‐E2. Seven (35%) babies with GBV‐C alone developed marginally elevated ALT; the baby with HCV and GBV‐C co‐infection had the highest ALT peak value (664 IU/l). Seven of the 141 (5%) babies born to the GBV‐C RNA negative mothers acquired HCV and six (85.7%) had abnormal ALT. The mean ALT peak value was significantly higher (P < 0.05) for babies with HCV than for those with GBV‐ C. None of the children with GBV‐C or with HCV became icteric. GBV‐C is frequently present in anti‐HCV positive women. The infection is transmitted efficiently from mother to baby and rate of transmission is much higher than that for HCV. GBV‐C can cause persistent infection in babies but usually without clear evidence of liver disease. J. Med. Virol. 54: 107–112, 1998.

Mycobacterium tuberculosis FurA Autoregulates Its Own Expression

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.

Pristinamycin-inducible gene regulation in mycobacteria

In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer. Three mycobacterial genes were cloned under pptr-Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be over-expressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin I. Thus it showed to be an effective inducible system in mycobacteria.

Bacteriophage P4 immunity controlled by small RNAs via transcription termination

Satellite bacteriophage P4 immunity is encoded within a short DNA region 357 bp long containing the promoter PLE and 275 bp downstream. PLE is active both in the early post‐infection phase, when genes necessary for P4 lytic cycle are transcribed from this promoter, and in the lysogenic condition, when expression of the above genes is prevented by prophage immunity.

Genetic analysis of the immunity region of phage‐plasmid P4

In the prophage P4, expression of the early genes is prevented by premature termination of transcription from the constitutive promoter Ple. In order to identify the region coding for the immunity determinant, we cloned several fragments of P4 DNA and tested their ability to confer immunity to P4 superinfection. A 357 bp long fragment (P4 8418‐8774) is sufficient to confer immunity to an infecting P4 phage and to complement the immunity‐defective P4 cl405 mutant, both in the presence and in the absence of the helper phage P2.

Genome-Wide Discovery of Small RNAs in Mycobacterium tuberculosis

Only few small RNAs (sRNAs) have been characterized in Mycobacterium tuberculosis and their role in regulatory networks is still poorly understood. Here we report a genome-wide characterization of sRNAs in M. tuberculosis integrating experimental and computational analyses. Global RNA-seq analysis of exponentially growing cultures of M. tuberculosis H37Rv had previously identified 1373 sRNA species. In the present report we show that 258 (19%) of these were also identified by microarray expression. This set included 22 intergenic sRNAs, 84 sRNAs mapping within 5′/3′ UTRs, and 152 antisense sRNAs. Analysis of promoter and terminator consensus sequences identified sigma A promoter consensus sequences for 121 sRNAs (47%), terminator consensus motifs for 22 sRNAs (8.5%), and both motifs for 35 sRNAs (14%). Additionally, 20/23 candidates were visualized by Northern blot analysis and 5′ end mapping by primer extension confirmed the RNA-seq data. We also used a computational approach utilizing functional enrichment to identify the pathways targeted by sRNA regulation. We found that antisense sRNAs preferentially regulated transcription of membrane-bound proteins. Genes putatively regulated by novel cis-encoded sRNAs were enriched for two-component systems and for functional pathways involved in hydrogen transport on the membrane.

Mycobacterium smegmatis RNase J is a 5-3 exo-/endoribonuclease and both RNase J and RNase E are involved in ribosomal RNA maturation

The presence of very different sets of enzymes, and in particular the presence of RNase E and RNase J, has been used to explain significant differences in RNA metabolism between the two model organisms Escherichia coli and Bacillus subtilis. However, these studies might have somewhat polarized our view of RNA metabolism. Here, we identified a RNase J in Mycobacterium smegmatis that has both 5′‐3′ exo‐ and endonucleolytic activity. This enzyme coexists with RNase E in this organism, a configuration that enabled us to study how these two key nucleases collaborate. We demonstrate that RNase E is responsible for the processing of the furA‐katG transcript in M. smegmatis and that both RNase E and RNase J are involved in the 5′ end processing of all ribosomal RNAs. In contrast to B. subtilis, the activity of RNase J, although required in vivo for 23S rRNA maturation, is not essential in M. smegmatis. We show that the pathways for ribosomal RNA maturation in M. smegmatis are quite different from those observed in E. coli and in B. subtilis. Studying organisms containing different combinations of key ribonucleases can thus significantly broaden our view of the possible strategies that exist to direct RNA metabolism.

WhiB5, a Transcriptional Regulator That Contributes to Mycobacterium tuberculosis Virulence and Reactivation

ABSTRACT The proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, a Mycobacterium tuberculosis protein belonging to this superfamily. A null mutant was constructed in M. tuberculosis H37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, the whiB5 mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain to S-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, including sigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.

Immobilized Biocatalysts for the Production of Nucleosides and Nucleoside Analogues by Enzymatic Transglycosylation Reactions

The recombinant enzymes uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) were over-expressed in high-biomass bacterial fermentations and co-immobilized, without previous purification, on epoxy-activated solid supports by covalent linkages. These preparations are efficient biocatalysts of transglycosylation reactions and have been developed for producting natural and modified nucleosides of pharmaceutical interest in the field of antiviral and antitumoral agents. The new biocatalysts described in this work are suitable for both laboratory and industrial scale applications due to the maintainance of high catalytic efficiency, thermal and solvent stability, reusability and ease of operation in batch as well as in continuous reactions.