pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola

The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5′-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgEⁱⁿ) regained the susceptibility to 5-FOA. Finally, FlgEⁱⁿ was plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgE^out) were obtained and characterized by PCR and Southern blotting analyses. The results showed that the flgE gene was deleted and FlgE^out was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE^out has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola.     

Isolation and characterisation of a linked cluster of genes from Agrobacterium tumefaciens encoding proteins involved in flagellar basal-body structure

We report the DNA sequence of 7205 bp of the Agrobacterium tumefaciens chromosome. This contains a putative operon encoding homologues of the flagellar rod and associated proteins FlgBCG and FliE, the L and P ring proteins (FlgHI) a possible flagellum-specific export protein FliP, and two proteins of unknown function, FlgA and FliL. Several of these genes have overlapping stop and start codons. Three non-flagellate Tn5-induced mutations map to this operon: fla-11 to the first gene, encoding the rod protein FlgB; fla-15 to flgA; and fla-12 to fliL. A site-specific mutation introduced into the final gene in this cluster, fliP, also resulted in a non-flagellate phenotype. This indicates that the operon is expressed, and that at least FlgB, FlgA, FliL and FliP are required for flagellar assembly in A. tumefaciens. The bulk of this operon is conserved in the same order in Rhizobium meliloti.     

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.Subject terms: Microbiology, Diseases     

FliZ Is a Global Regulatory Protein Affecting the Expression of Flagellar and Virulence Genes in Individual Xenorhabdus nematophila Bacterial Cells

Heterogeneity in the expression of various bacterial genes has been shown to result in the presence of individuals with different phenotypes within clonal bacterial populations. The genes specifying motility and flagellar functions are coordinately regulated and form a complex regulon, the flagellar regulon. Complex interplay has recently been demonstrated in the regulation of flagellar and virulence gene expression in many bacterial pathogens. We show here that FliZ, a DNA-binding protein, plays a key role in the insect pathogen, Xenorhabdus nematophila, affecting not only hemolysin production and virulence in insects, but efficient swimming motility. RNA-Seq analysis identified FliZ as a global regulatory protein controlling the expression of 278 Xenorhabdus genes either directly or indirectly. FliZ is required for the efficient expression of all flagellar genes, probably through its positive feedback loop, which controls expression of the flhDC operon, the master regulator of the flagellar circuit. FliZ also up- or downregulates the expression of numerous genes encoding non-flagellar proteins potentially involved in key steps of the Xenorhabdus lifecycle. Single-cell analysis revealed the bimodal expression of six identified markers of the FliZ regulon during exponential growth of the bacterial population. In addition, a combination of fluorescence-activated cell sorting and RT-qPCR quantification showed that this bimodality generated a mixed population of cells either expressing (“ON state”) or not expressing (“OFF state”) FliZ-dependent genes. Moreover, studies of a bacterial population exposed to a graded series of FliZ concentrations showed that FliZ functioned as a rheostat, controlling the rate of transition between the “OFF” and “ON” states in individuals. FliZ thus plays a key role in cell fate decisions, by transiently creating individuals with different potentials for motility and host interactions.     

The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions

Motility is a critical function needed for nutrient acquisition, biofilm formation, and the avoidance of harmful chemicals and predators. Flagellar motility is one of the most pressure-sensitive cellular processes in mesophilic bacteria; therefore, it is ecologically relevant to determine how deep-sea microbes have adapted their motility systems for functionality at depth. In this study, the motility of the deep-sea piezophilic bacterium Photobacterium profundum SS9 was investigated and compared with that of the related shallow-water piezosensitive strain Photobacterium profundum 3TCK, as well as that of the well-studied piezosensitive bacterium Escherichia coli. The SS9 genome contains two flagellar gene clusters: a polar flagellum gene cluster (PF) and a putative lateral flagellum gene cluster (LF). In-frame deletions were constructed in the two flagellin genes located within the PF cluster (flaA and flaC), the one flagellin gene located within the LF cluster (flaB), a component of a putative sodium-driven flagellar motor (motA2), and a component of a putative proton-driven flagellar motor (motA1). SS9 PF flaA, flaC, and motA2 mutants were defective in motility under all conditions tested. In contrast, the flaB and motA1 mutants were defective only under conditions of high pressure and high viscosity. flaB and motA1 gene expression was strongly induced by elevated pressure plus increased viscosity. Direct swimming velocity measurements were obtained using a high-pressure microscopic chamber, where increases in pressure resulted in a striking decrease in swimming velocity for E. coli and a gradual reduction for 3TCK which proceeded up to 120 MPa, while SS9 increased swimming velocity at 30 MPa and maintained motility up to a maximum pressure of 150 MPa. Our results indicate that P. profundum SS9 possesses two distinct flagellar systems, both of which have acquired dramatic adaptations for optimal functionality under high-pressure conditions.     

Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase

We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169° on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment. This fragment contained an incomplete and two complete open reading frames (ORFs). The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25 038 Da), was found to be responsible for the observed reduced mannitol fermentation. The 3′ part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identity. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72 889 Da. PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases. Several of the essential aa in the loops known to form the active site of cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA. After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue.     

Regulation of motility by the ExpR/Sin quorum-sensing system in Sinorhizobium meliloti

A successful symbiotic relationship between Sinorhizobium meliloti and its host Medicago sativa (alfalfa) depends on several signaling mechanisms, such as the biosynthesis of exopolysaccharides (EPS) by S. meliloti. Previous work in our laboratory has shown that a quorum-sensing mechanism controls the production of the symbiotically active EPS II. Recent microarray analysis of the whole-genome expression profile of S. meliloti reveals that the ExpR/Sin quorum-sensing system regulates additional physiological processes that include low-molecular-weight succinoglycan production, nitrogen utilization, metal transport, motility, and chemotaxis. Nearly half of the flagellar genes and their dependence on quorum sensing are prominently displayed in our microarray analyses. We extend those observations in this work and confirm the findings by real-time PCR expression analysis of selected genes, including the flaF, flbT, flaC, cheY1, and flgB genes, involved in motility and chemotaxis. These genes code for regulators of flagellum synthesis, the chemotactic response, or parts of the flagellar apparatus. Gene expression analyses and visualization of flagella by electron microscopy performed at different points in the growth phase support our proposed model in which quorum sensing downregulates motility in S. meliloti. We demonstrate that the ExpR/Sin quorum-sensing system controls motility gene expression through the VisN/VisR/Rem relay. We also show that the ExoS-dependent two-component system suppresses motility gene expression through VisN and Rem in parallel to quorum sensing. This study contributes to our understanding of the mechanisms that govern motility in S. meliloti.     

Sequencing and complementation analysis of the nifUSV genes from Azospirillum brasilense

The functionality of nitrogenase in diazotrophic bacteria is dependent upon nif genes other than the structural nifH, D, and K genes which encode the enzyme subunit proteins. Such genes are involved in the activation of nif gene expression, maturation of subunit proteins, cofactor biosynthesis, and electron transport. In this work, approximately 5500 base pairs located within the major nif gene cluster of Azospirillum brasilense Sp7 have been sequenced. The deduced open reading frames were compared to the nif gene products of Azotobacter vinelandii and other diazotrophs. This analysis indicates the presence of five ORFs encoding ORF2, nifU, nifS, nifV, and ORF4 in the same sequential organization as found in other organisms. Consensus σ⁵⁴ and NifA binding sites are present in the putative promoter region upstream of ORF2 in the A. brasilense sequence. The nifV gene of A. brasilense but not nifU or nifS complemented corresponding mutants strains of A. vinelandii.     

Molecular characterization of a flagellar/chemotaxis operon in the spirochete Borrelia burgdorferi

A chemotaxis gene cluster from Borrelia burgdorferi, the spirochete that causes Lyme disease, was cloned, sequenced, and analyzed. This cluster contained three chemotaxis gene homologs (cheA, cheW and cheY) and an open reading frame we identified as cheX. Although the major functional domains for B. burgdorferi CheW and CheY were well conserved, the size of cheW was significantly different from the homolog of other bacteria. Phylogenetic analysis of CheY indicated that B. burgdorferi constitutes a distinct branch with Treponema pallidum and is closely associated with Archea and Gram-positive bacteria. RT-PCR analysis indicated that the chemotaxis genes and the upstream flagellar gene flaA constitute an operon. Western blot analysis using antibody to Escherichia coli CheA resulted in two reactive proteins in the cell lysates of B. burgdorferi that is consistent with two cheA homologs being present in this organism. The results taken together suggest both similarities and differences in the chemotaxis apparatus of B. burgdorferi compared to those of other bacteria.     

Temporal and tissue specific gene expression patterns of the zebrafish kinesin-1 heavy chain family,kif5s,during development

Homo- and heterodimers of Kif5 proteins form the motor domain of Kinesin-1, a major plus-end directed microtubule motor. Kif5s have been implicated in the intracellular transport of organelles, vesicles, proteins, and RNAs in many cell types. There are three mammalian KIF5s. KIF5A and KIF5C proteins are strictly neural in mouse whereas, KIF5B is ubiquitously expressed. Mouse knockouts indicate crucial roles for KIF5 in development and human mutations in KIF5A lead to the neurodegenerative disease Hereditary Spastic Paraplegia. However, the developmental functions and the extent to which individual kif5 functions overlap have not been elucidated. Zebrafish possess five kif5 genes: kif5Aa, kif5Ab, kif5Ba, kif5Bb, and kif5C. Here we report their tissue specific expression patterns in embryonic and larval stages. Specifically, we find that kif5As are strictly zygotic and exhibit neural-specific expression. In contrast, kif5Bs exhibit strong maternal contribution and are ubiquitously expressed. Lastly, kif5C exhibits weak maternal expression followed by enrichment in neural populations. In addition, kif5s show distinct expression domains in the larval retina.     

A flagellar-specific ATPase (FliI) is necessary for flagellar export in Helicobacter pylori

Although flagellar motility is essential for the colonisation of the stomach by Helicobacter pylori, little is known about the regulation of flagellar biosynthesis in this organism. We have identified a gene in H. pylori, designated fliI, whose deduced amino acid sequence revealed extensive homology with the FliI/LcrB/InvC family of proteins which energise the export of flagellar and other virulence factors in several bacterial species. An isogenic mutant of fliI was non-motile and synthesised reduced amounts of flagellin and hook protein subunits. The majority (>99%) of mutant cells were completely aflagellate. These results suggest that FliI is a novel ATPase involved in flagellar export in H. pylori.