Michael J. Benedik

A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS.

SUMMARY Among bacterial toxin/antitoxin (TA) systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we demonstrate YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a novel TA system with YjdK (renamed GhoS) since in vitro RNA degradation studies, qRT-PCR, and whole-transcriptome studies revealed GhoS masks GhoT toxicity by cleaving specifically ghoT mRNA. Alanine substitutions showed arginine 28 is important for GhoS activity, and RNA sequencing indicated the GhoS cleavage site is rich in uridine and adenosine. The NMR structure of GhoS indicates it is related to the CAS2 CRISPR RNase, and GhoS is a monomer. Hence, GhoT/GhoS is the first type V TA system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.

Antitoxin MqsA helps mediate the bacterial general stress response

SUMMARY Although it is well-recognized that bacteria respond to environmental stress via global networks, the mechanism by which stress is relayed to the interior of the cell is poorly understood. Here we show that enigmatic toxin/antitoxin systems play a vital role in mediating the environmental stress response. Specifically, the antitoxin MqsA represses rpoS, which encodes the master regulator of stress. Repression of rpoS by MqsA reduces the concentration of the internal messenger 3,5-cyclic diguanylic acid, leading to increased motility and decreased biofilm formation. Furthermore, the repression of rpoS by MqsA decreases oxidative stress resistance via catalase activity. Upon oxidative stress, MqsA is rapidly degraded by Lon protease resulting in induction of rpoS. Hence, we show that external stress alters gene regulation controlled by toxin/antitoxin systems, such that the degradation of antitoxins during stress leads to a switch from the planktonic state (high motility) to the biofilm state (low motility).

Arrested Protein Synthesis Increases Persister-Like Cell Formation

ABSTRACT Biofilms are associated with a wide variety of bacterial infections and pose a serious problem in clinical medicine due to their inherent resilience to antibiotic treatment. Within biofilms, persister cells comprise a small bacterial subpopulation that exhibits multidrug tolerance to antibiotics without undergoing genetic change. The low frequency of persister cell formation makes it difficult to isolate and study persisters, and bacterial persistence is often attributed to a quiescent metabolic state induced by toxins that are regulated through toxin-antitoxin systems. Here we mimic toxins via chemical pretreatments to induce high levels of persistence (10 to 100%) from an initial population of 0.01%. Pretreatment of Escherichia coli with (i) rifampin, which halts transcription, (ii) tetracycline, which halts translation, and (iii) carbonyl cyanide m-chlorophenylhydrazone, which halts ATP synthesis, all increased persistence dramatically. Using these compounds, we demonstrate that bacterial persistence results from halted protein synthesis and from environmental cues.

Microbial nitrilases: versatile, spiral forming, industrial enzymes

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a αββα–αββα sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.

Microbial denitrogenation of fossil fuels

The microbial degradation of nitrogen compounds from fossil fuels is important because of the contribution these contaminants make to the formation of nitrogen oxides (NOx) and hence to air pollution and acid rain. They also contribute to catalyst poisoning during the refining of crude oil, thus reducing process yields. We review the current status of microbial degradation of aromatic nitrogen compounds and discuss the potential of microbial processes to alleviate these problems.

Induction of phospholipase- and flagellar synthesis in Serratia liquefaciens is controlled by expression of the flagellar master operon flhD.

When a liquid culture of Serratia spp. reaches the last part of the logarithmic phase of growth it induces the synthesis of several extracellular hydrolytic enzymes. In this communication we show that synthesis and secretion of the extracellular phospholipase is coupled to expression of flagella. Expression of flagella is demonstrated to follow a growth‐phase‐dependent pattern. Cloning, complementation studies and DNA‐sequencing analysis has identified a genetic region in Serratia liquefaciens which exhibits extensive homology to the Escherichia coli flhD flagellar master operon. Interruption of the chromosomal flhD operon in S. liquefaciens results in non‐flagellated and phospholipase‐negative cells, but the synthesis of other exoenzymes is not affected. By placing the flhD operon under the control of a foreign inducible promoter we have shown that increased transcription through the flhD operon leads to induction of flagellar synthesis and phospholipase expression.

The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli

We are studying exoproteins of the enteric bacterium Serratia marcescens as a model system for the release of extracellular proteins from the cell. In this work we report the cloning of the gene for a secreted nuclease from S. marcescens and its complete nucleotide sequence. Following expression of the nuclease gene in both S. marcescens and Escherichia coli we were able to demonstrate the presence of the nuclease extracellularly in both organisms. Cell lysis did not occur and there was no concurrent release of cytoplasmic or periplasmic proteins. No accessory genes appeared to be required for extracellular secretion of the nuclease from E. coli. We can conclude that E. coli is capable of secreting certain proteins extracellularly, and may be a suitable host organism for the genetic analysis of extracellular protein secretion when provided with a suitable protein to export.

Cadaverine Inhibition of Porin Plays a Role in Cell Survival at Acidic pH

When grown at acidic pH, Escherichia coli cells secrete cadaverine, a polyamine known to inhibit porin-mediated outer membrane permeability. In order to understand the physiological significance of cadaverine excretion and the inhibition of porins, we isolated an OmpC mutant that showed resistance to spermine during growth and polyamine-resistant porin-mediated fluxes. Here, we show that the addition of exogenous cadaverine allows wild-type cells to survive a 30-min exposure to pH 3.6 better than cells expressing the cadaverine-insensitive OmpC porin. Competition experiments between strains expressing either wild-type or mutant OmpC showed that the lack of sensitivity of the porin to cadaverine confers a survival disadvantage to the mutant cells at reduced pH. On the basis of these results, we propose that the inhibition of porins by excreted cadaverine represents a novel mechanism that provides bacterial cells with the ability to survive acid stress.

Bacterial persistence increases as environmental fitness decreases

Since persister cells cause chronic infections and since Escherichia coli toxin MqsR increases persisters, we used protein engineering to increase the toxicity of MqsR to gain insights into persister cell formation. Through two amino acid replacements that increased the stability of MqsR, toxicity and persistence were increased. A whole‐transcriptome study revealed that the MqsR variant increased persistence by repressing genes for acid resistance, multidrug resistance and osmotic resistance. Corroborating these microarray results, deletion of rpoS, as well as the genes that the master stress response regulator RpoS controls, increased persister formation dramatically to the extent that nearly the whole population became persistent. Furthermore, wild‐type cells stressed by prior treatment to acid or hydrogen peroxide increased persistence 12 000‐fold. Whole‐transcriptome analyses of persister cells generated by two different methods (wild‐type cells pretreated with hydrogen peroxide and the rpoS deletion) corroborated the importance of suppressing RpoS in persister cell formation. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS‐based regulation of acid resistance, multidrug resistance and osmotic resistance systems.

CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies.

ABSTRACT The cyanide dihydratase in Bacillus pumilus was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in Escherichia coli. Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has Km and Vmax values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative Pseudomonas stutzeri strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of B. pumilus (strain 8A3) was cloned and expressed. This suggests that cynD, the gene coding for the cyanide dihydratase, is not unique to the C1 strain of B. pumilus and is not a reflection of its origin at a mining waste site.