Lipopolysaccharide of the Complex.

Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host's innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.

Inhibitors of bacterial HS biogenesis targeting antibiotic resistance and tolerance.

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (HS)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of HS in two major human pathogens, and , and discovered small molecules that inhibit bacterial CSE.

CydDC functions as a cytoplasmic cystine reductase to sensitize to oxidative stress and aminoglycosides.

l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in the component of cytochrome -I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis.

HS, a Bacterial Defense Mechanism against the Host Immune Response.

The biological mediator hydrogen sulfide (HS) is produced by bacteria and has been shown to be cytoprotective against oxidative stress and to increase the sensitivity of various bacteria to a range of antibiotic drugs. Here we evaluated whether bacterial HS provides resistance against the immune response, using two bacterial species that are common sources of nosocomial infections, and Elevations in HS levels increased the resistance of both species to immune-mediated killing. Clearances of infections with wild-type and genetically HS-deficient and were compared and in mouse models of abdominal sepsis and burn wound infection.

Mechanism of HS-mediated protection against oxidative stress in .

Endogenous hydrogen sulfide (HS) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous HS in Cellular resistance to HO strongly depends on the activity of , a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders ∆ cells hypersensitive to HO Conversely, induction of chromosomal from a strong pLtetO-1 promoter (P -) renders ∆ cells fully resistant to HO Furthermore, the endogenous level of HS is reduced in ∆ or ∆ ∆ cells but restored after the addition of an iron chelator dipyridyl.

S-nitrosylation of peroxiredoxin 1 contributes to viability of lung epithelial cells during Bacillus anthracis infection.

Background: Using Bacillus anthracis as a model gram-positive bacterium, we investigated the effects of host protein S-nitrosylation during bacterial infection. B. anthracis possesses a bacterial nitric oxide synthase (bNOS) that is important for its virulence and survival.

Glutamine versus ammonia utilization in the NAD synthetase family.

NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS). Members of the NADS family are present in nearly all species across the three kingdoms of Life.

H2S: a universal defense against antibiotics in bacteria.

Many prokaryotic species generate hydrogen sulfide (H(2)S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine β-synthase, cystathionine γ-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H(2)S production, rendering these pathogens highly sensitive to a multitude of antibiotics.

Linking RNA polymerase backtracking to genome instability in E. coli.

Frequent codirectional collisions between the replisome and RNA polymerase (RNAP) are inevitable because the rate of replication is much faster than that of transcription. Here we show that, in E. coli, the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs).

Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics.

Bacterial nitric oxide synthases (bNOS) are present in many Gram-positive species and have been demonstrated to synthesize NO from arginine in vitro and in vivo. However, the physiological role of bNOS remains largely unknown. We show that NO generated by bNOS increases the resistance of bacteria to a broad spectrum of antibiotics, enabling the bacteria to survive and share habitats with antibiotic-producing microorganisms.

Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages.

Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen-Bacillus anthracis-critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages.

Monitoring of gene knockouts: genome-wide profiling of conditionally essential genes.

We have developed a new microarray-based genetic technique, named MGK (Monitoring of Gene Knockouts), for genome-wide identification of conditionally essential genes. MGK identified bacterial genes that are critical for fitness in the absence of aromatic amino acids, and was further applied to identify genes whose inactivation causes bacterial cell death upon exposure to the bacteriostatic antibiotic chloramphenicol. Our findings suggest that MGK can serve as a robust tool in functional genomics studies.

Comparative genomics of NAD biosynthesis in cyanobacteria.

Biosynthesis of NAD(P) cofactors is of special importance for cyanobacteria due to their role in photosynthesis and respiration. Despite significant progress in understanding NAD(P) biosynthetic machinery in some model organisms, relatively little is known about its implementation in cyanobacteria. We addressed this problem by a combination of comparative genome analysis with verification experiments in the model system of Synechocystis sp.

Efficient gene inactivation in Bacillus anthracis.

A procedure for high-efficiency gene inactivation in Bacillus anthracis has been developed. It is based on a highly temperature-sensitive plasmid vector carrying kanamycin resistance cassette surrounded by DNA fragments flanking the desired insertion site. The approach was tested by constructing glutamate racemase E1 (racE1), glutamate racemase E2 (racE2) and comEC knock-out mutants of B.

Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria.

Thiamin and riboflavin are precursors of essential coenzymes-thiamin pyrophosphate (TPP) and flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD), respectively. In Bacillus spp, genes responsible for thiamin and riboflavin biosynthesis are organized in tightly controllable operons. Here, we demonstrate that the feedback regulation of riboflavin and thiamin genes relies on a novel transcription attenuation mechanism.

From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets.

Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase.

Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Rault, M.

[The production of recombinant polypeptides from the herpes simplex viruses types 1 and 2].

The genes IE175, IE63, IE68, IE12, UL29 and UL19 of herpes simplex virus types 1 and 2 were cloned. Fragments of these genes were cloned into open reading frame expression vector pEX1-3. Recombinant proteins containing amino acid sequences of ICP4, ICP27, ICP22, ICP47, major DNA-binding protein and major capsid protein were expressed in E.

[Cloning the human cytomegalovirus immediate-early gene and expression of it in Escherichia coli].

The major immediate early gene of human cytomegalovirus has been cloned. The fragment of the immediate early protein containing 351 amino acids has been fused with cro-beta-galactosidase and expressed in Escherichia coli cells. The obtained recombinant protein reacts in immunoblotting with the antibodies in human sera from patients suffering from acute cytomegalovirus infection.

[Cloning and expression of certain herpes simplex virus type 1 genes in Escherichia coli].

Complete genes IE12, IE63, IE68, IE175, UL19, UL29 of herpes simplex virus type I or their fragments have been cloned in Escherichia coli cells. The peptides expressed were shown to be fused with cro-beta-galactosidase proteins. The recombinant proteins containing amino acid sequences of ICP4, ICP27 and ICP47, major capsid protein, and major DNA-binding protein react in immunoblotting with the anti-HSVI serum from hyperimmune rabbit.

[The determination of the antigenic activity of recombinant virus-specific polypeptides from the herpes simplex virus types 1 and 2 and their use in immunoenzyme analysis].

The structural and nonstructural recombinant proteins of HSV type 1 and type 2 were expressed in E. coli by fusion with cro-beta-galactosidase proteins. These proteins containing amino acid sequences of ICP4, ICP27, ICP47, ICP22, major capsid protein and major DNA-binding proteins of HSV types 1 and 2 reacted in immunoblotting with the corresponding anti-HSV sera from hyperimmune rabbits.

[Analysis of insertions of the determinant of drug resistance to kanamycin and chloramphenicol into bacteriophage lambda att80].

The insertion sites of elements Tn9 and Tn601 which determine chloramphenicol and kanamycin resistance have been detected restriction analysis. The functioning of transposons i.e.

[Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location].

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb.

[Genetic control of plasmid F' formation. II. Role of recipient recA- in the process of plasmid F' formation].

We have chosen the process of F'-plasmid formation as a model for a study of the illegitimate, recA-independent recombination. Our genetic data showed that F'-formation was under genetic control of the Hfr donor cells. To determine the role of recipient cells in the process of F-prime formation we compared the pattern of transconjugant formation in the crosses of Hfr and F' donors with the recA-nalA+ and recA-nalA- recipients, nalA- mutation in the recipient lead to the sharp decrease of the total yield of transconjugants selected for proximal markers and decrease the fraction of conjugative plasmids in progeny.