Indirect induction of SOS functions in Salmonella typhimurium

Infection of non-UV-irradiated cells of Salmonella typhimurium with UV-damaged P22 or KB1 phage induces recA-dependent inhibition of cell division, cell mutagenesis and prophage induction but not inhibition of respiration. On the contrary, respiration and ATP concentration are increased after treatment with UV-damaged phage in both RecA+ and RecA- strains, showing that this increase is not recA-dependent. Furthermore, infection with UV-damaged phage prevents both inhibition of respiration and decrease in ATP level in the UV-irradiated RecA+ strain. This indirect induction of SOS functions is related to degradation of phage DNA as well as to the multiplicity of infection used, suggesting that DNA degradation may play an important role in the mechanism of expression of the SOS system. Our results give also support to the hypothesis that there exists a differentiation in the expression of the various SOS functions.     

Inhibition of Phage Growth by an Antibiotic Rugulosin Isolated from Myrothecium verucaria

It was found that (?)rugulosin, an antibiotic isolated from Myrothecium verucaria, had a potent anti-phage effect on RNA phages (MS2, GA and Qβ) and DNA phages (δA and T4). The effect was almost independent of the host bacterial strains used. By a detailed investigation using MS2, it was revealed that the antibiotic did not affect the free phage or host bacterium alone but inhibited phage multiplication, and the degree of the inhibition depended on the multiplicity of infection. The inhibition was not mainly due to a drop in the burst size but rather was due to a decrease of the phage-producing cells during the early stages of phage infection and replication.     

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA₃) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.     

Host genetic requirements for DNA release of lactococcal phage TP901‐1

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram‐positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901‐1 due to mutations that affect TP901‐1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three‐component glycosylation system (TGS) was shown to be required for TP901‐1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope‐associated component that triggers TP901‐1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram‐positive infecting phage.

We present a comparative genome analysis of three mutants of Lactococcus cremoris 3107 which are resistant to phage TP901‐1 due to mutations that affect its DNA release. Through genetic complementation and phage infection assays, we identified a novel lactococcal three‐component glycosylation system required for TP901‐1 infection. We provide new insights into the mostly unknown DNA release stage of a Gram‐positive phage, since glycosylation has not been implicated in such a process to date.     

The stability of toxigenicity in Clostridium botulinum types C and D.

Several type C and D strains of Clostridium botulinum, which had been converted to the toxgenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum. Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage. However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion. When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage. Filtrates of the supernatants of culture fluids of strains transferred without anti-phase serum converted non-toxigenic strains to toxigenicity at varying rates.     

Analysis of productivity in lysis-deficient lambda expression systems

The integrated state of lambda in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on lambda expression systems(21,22) have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of lambda systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential lambda protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector lambdaZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in lambda systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of lambda vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems. (c) 1992 John Wiley & Sons, Inc.