Systematic Dissection and Trajectory-Scanning Mutagenesis of the Molecular Interface That Ensures Specificity of Two-Component Signaling Pathways

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk

Horizontally acquired glycosyltransferase operons drive salmonellae lipopolysaccharide diversity.

The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions

Alternative Sigma Factor σH Modulates Prophage Integration and Excision in Staphylococcus aureus

The prophage is one of the most important components of variable regions in bacterial genomes. Some prophages carry additional genes that may enhance the toxicity and survival ability of their host bacteria. This phenomenon is predominant in Staphylococcus aureus, a very common human pathogen. Bioinformatics analysis of several staphylococcal prophages revealed a highly conserved 40-bp untranslated region upstream of the int gene. A small transcript encoding phage integrase was identified to be initiated from the region, demonstrating that the untranslated region contained a promoter for int. No typical recognition sequence for either σA or σB was identified in the 40-bp region. Experiments both in vitro and in vivo demonstrated that σH recognized the promoter and directed transcription. Genetic deletion of sigH altered the int expression, and subsequently, the excision proportion of prophage DNAs. Phage assays further showed that sigH affected the ability of spontaneous lysis and lysogenization in S. aureus, suggesting that sigH plays a role in stabilizing the lysogenic state. These findings revealed a novel mechanism of prophage integration specifically regulated by a host-source alternative sigma factor. This mechanism suggests a co-evolution strategy of staphylococcal prophages and their host bacteria

Regulation of the Escherichia coli HipBA Toxin-Antitoxin System by Proteolysis

Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon- background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence

Replication and active partition of integrative and conjugative elements (ICEs) of the SXT/R391 family : the line between ICEs and conjugative plasmids is getting thinner

Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells

Role and Regulation of Osmolytes and ABA Interaction in Salt and Drought Stress Tolerance

Abiotic stress conditions lead to the defects in plant growth and development and also reduction in flowering and fertility. Under prolonged stresses, imminent death of the plants has been observed. To cope with such stress conditions, plants accumulate a wide variety of organic solutes called osmolytes. Osmolytes are accumulated in bacteria, lower, and higher plants as a response primarily to abiotic stress. They encompass amino acids such as proline, tertiary sulfonium, and quaternary ammonium compounds like beatines, sugars (trehalose), and polyhydric alcohols (mannitol, sorbitol, pinitol, etc.). Osmolytes are accumulated in the cytoplasm as well as in chloroplasts in certain cases for osmotic adjustment under stress conditions. This enables the plants to absorb water and survive under stress. Out of the many phytohormones that play diverse roles during abiotic stress, abscisic acid (ABA) is an important one and perceived by plants by a core signaling module. As an integral part of signal transduction during stress conditions, ABA and other hormones regulate not only stomatal closure, but also a wide array of gene expressions including osmolyte biosynthetic pathway genes. Many signal molecules like nitric oxide, carbon monoxide, and hydrogen sulfide also play a vital role in osmolyte biosynthesis. Osmolytes appear to have multiple functions during stress such as osmotic adjustment and scavenging of reactive oxygen species (ROS). Thus, generation of ROS and osmolyte accumulation are linked together. This review summarizes the role played by ABA in signal transduction, the role of hormones to regulate osmolyte biosynthesis, and various functions carried out by them