Joyce E. Karlinsey

Completion of the hook–basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription

The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline‐inducible promoter PtetA using the T‐POP transposon. Cells containing this construct are motile in the presence of tetracycline and non‐motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti‐sigma factor in the growth medium. This also coincides with the completion of hook–basal body structures. Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the hook–basal body structure.

Identification of New Flagellar Genes of Salmonella enterica Serovar Typhimurium

RNA levels of flagellar genes in eight different genetic backgrounds were compared to that of the wild type by DNA microarray analysis. Cluster analysis identified new, potential flagellar genes, three putative methyl-accepting chemotaxis proteins, STM3138 (McpA), STM3152 (McpB), and STM3216(McpC), and a CheV homolog, STM2314, in Salmonella, that are not found in Escherichia coli. Isolation and characterization of Mud-lac insertions in cheV, mcpB, mcpC, and the previously uncharacterized aer locus of S. enterica serovar Typhimurium revealed them to be controlled by sigma28-dependent flagellar class 3 promoters. In addition, the srfABC operon previously isolated as an SsrB-regulated operon clustered with the flagellar class 2 operon and was determined to be under FlhDC control. The previously unclassified fliB gene, encoding flagellin methylase, clustered as a class 2 gene, which was verified using reporter fusions, and the fliB transcriptional start site was identified by primer extension analysis. RNA levels of all flagellar genes were elevated in flgM or fliT null strains. RNA levels of class 3 flagellar genes were elevated in a fliS null strain, while deletion of the fliY, fliZ, or flk gene did not affect flagellar RNA levels relative to those of the wild type. The cafA (RNase G) and yhjH genes clustered with flagellar class 3 transcribed genes. Null alleles in cheV, mcpA, mcpB, mcpC, and srfB did not affect motility, while deletion of yhjH did result in reduced motility compared to that of the wild type.

Translation/Secretion Coupling by Type III Secretion Systems

Type III secretion systems mediate export of virulence proteins and flagellar assembly subunits in Gram-negative bacteria. Chaperones specific to each class of secreted protein are believed to prevent degradation of the secreted substrates. We show that an additional role of chaperones may be to regulate translation of secreted proteins. We show that the chaperone FIgN is required for translation of the flgM gene transcribed from one mRNA transcript (a flagellar class 3 transcript), but not from another (a flagellar class 2 transcript). FIgM translated from the class 3 transcript is primarily secreted whereas FIgM translated from the class 2 transcript is primarily retained in the cytoplasm. These results suggest FIgM and other type III secretion substrates possess both mRNA and amino acid secretion signals, and supports a new role for type III chaperones in translation/secretion coupling.

Biosynthesis and IroC‐dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium

In response to iron deprivation, Salmonella enterica serovar Typhimurium secretes two catecholate‐type siderophores, enterobactin and its glucosylated derivative salmochelin. Although the systems responsible for enterobactin synthesis and acquisition are well characterized, the mechanisms of salmochelin secretion and acquisition, as well as its role in Salmonella virulence, are incompletely understood. Herein we show by liquid chromatography‐mass spectrometry analysis of culture supernatants from wild type and isogenic mutant bacterial strains that the Major Facilitator Superfamily pump EntS is the major exporter of enterobactin and the ABC transporter IroC exports both salmochelin and enterobactin. Growth promotion experiments demonstrate that IroC is not required for utilization of Fe‐enterobactin or Fe‐salmochelin, as had been previously suggested, but the ABC transporter protein FepD is required for utilization of both siderophores. Salmonella mutants deficient in salmochelin synthesis or secretion exhibit reduced virulence during systemic infection of mice.

Humanized nonobese diabetic-scid IL2rγnull mice are susceptible to lethal Salmonella Typhi infection

Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S. Typhi can replicate in vivo in nonobese diabetic (NOD)-scid IL2rγnull mice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast, S. Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknown Salmonella virulence determinants, including Salmonella Pathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication of S. Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights into S. Typhi pathogenesis and devise strategies for the prevention of typhoid fever.

λ‐Red Genetic Engineering in Salmonella enterica serovar Typhimurium

Abstract The use of the recombination system from bacteriophage lambda, λ‐Red, allows for PCR‐generated fragments to be targeted to specific chromosomal locations in sequenced genomes. A minimal region of homology of 30 to 50 bases flanking the fragment to be inserted is all that is required for targeted mutagenesis. Procedures for creating specific insertions, deletions, and site‐directed changes are described.

The type III secretion chaperone FlgN regulates flagellar assembly via a negative feedback loop containing its chaperone substrates FlgK and FlgL.

The type III secretion (TTS) chaperones are small proteins that act either as cytoplasmic bodyguards, protecting their secretion substrates from degradation and aggregation, facilitators of their cognate substrate secretion or both. FlgN has been previously shown to be a TTS chaperone for the hook‐associated proteins FlgK and FlgL (FlgKL), and a translational regulator of the anti‐σ28 factor FlgM. Protein stability assays indicate that a flgN mutation leads to a dramatic decrease in the half‐life of intracellular FlgK. However, using gene reporter fusions to flgK we show that a flgN mutation does not affect the translation of a flgK–lacZ fusion. Quantification of FlgM protein levels showed that FlgKL inhibit the positive regulation on flgM translation by FlgN when secretion of FlgKL is inhibited. Suppressors of the motility‐defective phenotype of a flgN mutant were isolated and mapped to the clpXP and fliDST loci. Overexpression of flgKL on a plasmid also suppressed the motility defect of a flgN null mutant. These results suggest that FlgN is not required for secretion of FlgKL and that FlgN typifies a class of TTS chaperones that allows for the minimal amount of their substrates expression required in the assembly process by protecting the substrate from proteolysis. Our data leads us to propose a model in which the interaction between FlgN and FlgK or FlgL is a sensing mechanism to determine the stage of flagellar assembly. Furthermore, the interaction between FlgN and FlgK or FlgL inhibits the translational regulation of flgM via FlgN in response to the stage of flagellar assembly.

FliK regulates flagellar hook length as an internal ruler

The mechanism of length control of the flagellar hook is under debate between two theories. One claims that the FliK directly measures the hook length as a molecular ruler, while the other claims that the cytoplasmic substructure measures the amount of hook subunits to determine the hook length. Both agree that the FliK C‐terminal domain catalyses the substrate‐specificity switch to terminate hook elongation. In this study, we systematically created fliK mutants with deletions and insertions at various sites within the FliK N‐terminal domain and analysed their effects on the final hook length. Insertions of peptide fragments from the Yersinia YscP into FliK gave rise to hooks with defined lengths, which was proportional to the molecular size of the FliK‐YscP chimeras. Among fliK deletion mutants, only those with small truncations in three specific sites of FliK produced hooks of a defined, shortened length. For the majority of deletion mutants, FliK was secreted, but hook length was not controlled. On the other hand, for some deletion mutants FliK was not secreted, but the hook length was controlled, indicating that FliK secretion is not necessary for hook‐length control. We conclude that FliK regulates hook length as an internal molecular ruler.

The phage shock protein PspA facilitates divalent metal transport and is required for virulence of Salmonella enterica sv. Typhimurium

The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ityr) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance‐associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp‐homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn2+ transport by MntH and SitABCD, as well as Zn2+ and Mn2+ transport by ZupT. In vitro uptake of 54Mn2+ by MntH and ZupT was reduced in the absence of PspA. Transport‐deficient mutants exhibit reduced viability in the absence of PspA when grown under metal‐limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1‐expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection.

Simultaneous purification of DNA and RNA from small numbers of eukaryotic cells

An extraction procedure for the simultaneous isolation of RNA and DNA from tissue culture cells is described. The procedure is a variation of the guanidium/lithium chloride method for RNA isolation which is rapid, simple, and avoids costly ultracentrifugation equipment. The genomic DNA yielded by this procedure is greater than 50 kb in length and may be readily cleaved by restriction endonucleases. Sufficient DNA for Southern blot analysis, and RNA for Northern blot or nuclease protection analysis, can be obtained from as few as 2 x 10(6) cells, making this method particularly suitable for the genetic screening of large numbers of individual, stably transfected cell clones.