Bacteria sense and respond to a wide range of physical and chemical signals. Central to sensing and responding to these signals are two-component systems, which have a sensor histidine kinase (SK) and a response regulator (RR) as basic components. Here we review the different molecular mechanisms by which these signals are integrated and modulate the phosphorylation state of SKs. Apart from the basic mechanism, which consists of signal recognition by the SK that leads to an alteration of its autokinase activity and subsequently a change in the RR phosphorylation state, a variety of alternative modes have evolved. The biochemical data available on SKs, particularly their molecular interactions with signals, nucleotides, and their cognate RRs, are also reviewed.

Bacterial Sensor Kinases: Diversity in the Recognition of Environmental Signals

The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application.

/pdf/learning-from-bacteriophages-advantages-and-limitations-of-1le5ap5501.pdf

Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications

Viruses are powerful tools for investigating and manipulating their hosts, but the enormous size and amazing genetic diversity of the bacteriophage population have emerged as something of a surprise. In light of the evident importance of mycobacteria to human health--especially Mycobacterium tuberculosis, which causes tuberculosis--and the difficulties that have plagued their genetic manipulation, mycobacteriophages are especially appealing subjects for discovery, genomic characterization, and manipulation. With more than 70 complete genome sequences available, the mycobacteriophages have provided a wealth of information on the diversity of phages that infect a common bacterial host, revealed the pervasively mosaic nature of phage genome architectures, and identified a huge number of genes of unknown function. Mycobacteriophages have provided key tools for tuberculosis genetics, and new methods for simple construction of mycobacteriophage recombinants will facilitate postgenomic explorations into mycobacteriophage biology.

Mycobacteriophages: Genes and Genomes

The study of mycobacteriophages provides insights into viral diversity and evolution, as well as the genetics and physiology of their pathogenic hosts. Genomic characterization of 80 mycobacteriophages reveals a high degree of genetic diversity and an especially rich reservoir of interesting genes. These include a vast number of genes of unknown function that do not match known database entries and many genes whose functions can be predicted but which are not typically found as components of phage genomes. Thus many mysteries surround these genomes, such as why the genes are there, what do they do, how are they expressed and regulated, how do they influence the physiology of the host bacterium, and what forces of evolution directed them to their genomic homes? Although the genetic diversity and novelty of these phages is full of intrigue, it is a godsend for the mycobacterial geneticist, presenting an abundantly rich toolbox that can be exploited to devise new and effective ways for understanding the genetics and physiology of human tuberculosis. As the number of sequenced genomes continues to grow, their mysteries continue to thicken, and the time has come to learn more about the secret lives of mycobacteriophages.

The secret lives of mycobacteriophages.

Phage-mediated metabolic changes in bacteria are hypothesized to markedly alter global nutrient and biogeochemical cycles. Despite their theoretic importance, experimental data on the net metabolic impact of phage infection on the bacterial metabolism remains scarce. In this study, we tracked the dynamics of intracellular metabolites using untargeted high coverage metabolomics in Pseudomonas aeruginosa cells infected with lytic bacteriophages from six distinct phage genera. Analysis of the metabolomics data indicates an active interference in the host metabolism. In general, phages elicit an increase in pyrimidine and nucleotide sugar metabolism. Furthermore, clear phage-specific and infection stage-specific responses are observed, ranging from extreme metabolite depletion (for example, phage YuA) to complete reorganization of the metabolism (for example, phage phiKZ). As expected, pathways targeted by the phage-encoded auxiliary metabolic genes (AMGs) were enriched among the metabolites changing during infection. The effect on pyrimidine metabolism of phages encoding AMGs capable of host genome degradation (for example, YuA and LUZ19) was distinct from those lacking nuclease-encoding genes (for example, phiKZ), which demonstrates the link between the encoded set of AMGs of a phage and its impact on host physiology. However, a large fraction of the profound effect on host metabolism could not be attributed to the phage-encoded AMGs. We suggest a potentially crucial role for small, 'non-enzymatic' peptides in metabolism take-over and hypothesize on potential biotechnical applications for such peptides. The highly phage-specific nature of the metabolic impact emphasizes the potential importance of the 'phage diversity' parameter when studying metabolic interactions in complex communities.

/pdf/high-coverage-metabolomics-analysis-reveals-phage-specific-3n917exuga.pdf

High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection

st proline residue of the wild- type repressor was changed to leucine in the ts mutant repressor. The 100% identity that was discovered between the two DNA regions of phages L1 and L5, carrying the same sets of genes including their repressor genes, strengthened the speculation that L1 is a minor variant of phage L5 or vice versa. A comparative analysis of the repressor proteins of different mycobacteriophages suggests that the mycobacteriophage-specific repressor proteins constitute a new family of repressors, which were possibly evolved from a common ancestor. Alignment of the mycobacteriophage-specific repressor proteins showed at least 7 blocks (designated I-VII) that carried 3-8 identical amino acid residues. The amino acid residues of blocks V, VI, and some residues downstream to block VI are crucial for the function of the L1 (or L5) repressor. Blocks I and II possibly form the turn and helix 2 regions of the HTH motif of the repressor. Block IV in the L1 repressor is part of the most charged region encompassing amino acid residues 72-92, which flanks the putative N- terminal basic (residues 1-71) and C-terminal acidic (residues 93-183) domains of L1 repressor.

/pdf/cloning-and-sequencing-analysis-of-the-repressor-gene-of-2jvhukrx6a.pdf

Cloning and sequencing analysis of the repressor gene of temperate mycobacteriophage L1.

Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery.

Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

Earlier, we reported that the bacteriophage lambda P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this lambda P gene-mediated lethality. In this paper, we show that under the lambda P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94 % linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from lambda P gene-mediated killing and complements E. coli dnaAts46 at 42 degrees C. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to lambda P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.

The Mutation that Makes Escherichia coli Resistant to λ P Gene-mediated Host Lethality Is Located within the DNA Initiator Gene dnaA of the Bacterium

A deoxyriboendonuclease has been purified to near homogeneity from a fast growing mycobacterium species, M. smegmatis and characterized to some extent. The size of enzyme is about 43 kDa as determined by a denaturing gel analysis. It shows optimum activity at 32 degrees C in Tris-HCl buffer (pH 7.2) containing 2.5 mM of MgCl2. Both EDTA and K+ but not Na+ inhibit its activity. Evidences show that the enzyme is not a restriction endonuclease but catalyzes the endonucleolytic cleavage of both the double- as well as the single-strand DNA non-specifically. It has been shown that the cleavage by this enzyme generates DNA fragments carrying phosphate groups at 5' ends and hydroxyl group at the 3' ends, respectively. Analysis reveals that no endonuclease having size and property identical to our deoxyriboendonuclease had been purified from M. smegmatis before. The property of our enzymes closely matches with the deoxyriboendonucleases purified from diverse sources including bacteria.

Purification and characterization of a deoxyriboendonuclease from Mycobacterium smegmatis.

To get clues about the genes as well as the gene regulatory circuit controlling the lytic development of temperate mycobacteriophage L1, previously we screened several conditional lethal mutants of L1 and characterized some of them to an extent. One of the mutants, L1G23ts23, was found defective in both growth and late gene transcription at 42∞C but not at 32∞C. Here we show that the above phage mutant is also defective in the expression of phage-coded deoxyribonuclease (DNase) at 42∞C but not at 32∞C. The G23 gene however does not code for the above enzyme. Further analyses using the L1G23ts23 mutant suggest that synthesis of DNase is also not regulated by G23 at transcriptional level. Expression of functional DNase in fact requires de novo protein synthesis. Among the 25 revertants isolated from the L1G23ts23 mutant, which are capable of growing at 42∞C (by overcoming the ts defect in late transcription), two, R4 and R22, have been shown to retain the ts defect in the expression of the above enzyme and R4, to retain also the G23ts23 mutation. This suggests that R4 (R22 was not tested for the presence of G23ts23 mutation) carries an extragenic suppressor of G23ts23 mutation in a different gene (we call this putative gene as Gx), which now helps bypass the requirement of G23 for late gene transcription. Possible role of G23 on the regulation of L1-coded Gx and deoxyribonuclease has been discussed at length.

The Delayed Early Gene G23 of Temperate Mycobacteriophage L1 Regulates the Expression of Deoxyribonuclease, the Product of another Delayed Early Gene of the Phage

Nitai Chandra Mandal

Papers

Cloning and sequencing analysis of the repressor gene of temperate mycobacteriophage L1.

Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

The Mutation that Makes Escherichia coli Resistant to λ P Gene-mediated Host Lethality Is Located within the DNA Initiator Gene dnaA of the Bacterium

Purification and characterization of a deoxyriboendonuclease from Mycobacterium smegmatis.

The Delayed Early Gene G23 of Temperate Mycobacteriophage L1 Regulates the Expression of Deoxyribonuclease, the Product of another Delayed Early Gene of the Phage