- Identify different ways to run motility testing
SIM deeps are a multi-test medium comprising 3 tests: sulfide (H2S gas), indole production, and motility. You have already tested for motility via the hanging drop slide, and here is an additional way to determine it, with less muss and fuss. In fact, there is an even better way to determine motility, motility agar with tetrazolium dye, that you will also be using in lab. Both of these agars have advantages. SIM is actually 3 tests in one, but, on the other hand, the motility with tetrazolium is much easier to read for motility. The tetrazolium is a colorless salt which becomes red when reduced, occurring as a result of bacterial metabolism.
Mitochondrial reduction of MTT to blue formazan product from Wikipedia (credit: Rogan Grant).
- SIM agar deep per organism
- 1 TTC motility agar with tetrazolium dye per organism
- Organisms: Klebsiella, E. coli
- Inoculate into a tube of SIM media with a NEEDLE, all the way to the bottom.
- Inoculate into a tube of TTC motility agar with tetrazolium dye with a NEEDLE, all the way to the bottom.
- Incubate at 25 or 37 degrees C.
- Hold the tube up to the light and look at the stab line to determine motility.
- If NONMOTILE, you will see the intact straight stab line.
- If MOTILE, the original stab line will diffuse out into the medium as the bacteria spread throughout. In fact, you may not even be able to see a stab line at all: it may be turbid throughout the medium. FOR THAT REASON, you might want to compare the inoculated SIM with an uninoculated one.
Motility agar with tetrazolium
The same description from above applies to this medium, BUT with the added, helpful colored dye tetrazolium which turns red as a result of the bacteria metabolizing.
- What is the function of tetrazolium?
- How do you tell if the organism is motile?
The Triple Sugar Iron (TSI) Test – Principle, Procedure, Uses and Interpretation
Most bacteria have the ability to ferment carbohydrates, particularly sugars. Among them, each bacteria can ferment only some of the sugars, while it cannot ferment the others. Thus, the sugars, which a bacteria can ferment and the sugars, which it cannot is the characteristic of the bacteria and thus an important criterion for its identification.
The Triple Sugar Iron (TSI) test is a microbiological test named for its ability to test a microorganism’s ability to ferment sugars and to produce hydrogen sulfide. An agar slant of a special medium with multiple sugars constituting a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate is used for carrying out the test. All of these ingredients when mixed together and allowed solidification at an angle result in a agar test tube at a slanted angle. The slanted shape of this medium provides an array of surfaces that are either exposed to oxygen-containing air in varying degrees (an aerobic environment) or not exposed to air (an anaerobic environment) under which fermentation patterns of organisms are determined.
1. Wet Mount 2. The Tube Motility Test
1. Make suspension of a colony of test organisms in distilled water on a glass slide. Alternatively, a loop of medium from a fresh broth culture can be used. Put a cover glass on it. Examine under the microscope using hypochlorite solution as it contains live organisms.
2. Hanging Drop Method: Clean a cover slip. Apply Vaseline at its 4 corners. Put a drop of distilled water in the center and emulsify a colony of test organisms. Put the glass slide gently on it and hold it up side down. See under microscope with 10X and then 40X objective. Margins of drops are specifically seen. Motile organisms are clearly seen moving rapidly in the field. Non-motile organisms show to-and-fro Brownian motion, but these don’t move in relation to each other.
Novel Single-Tube Agar-Based Test System for Motility Enhancement and Immunocapture of Escherichia coli O157:H7 by H7 Flagellar Antigen-Specific AntibodiesFIG. 1 . Schematic showing preparation and inoculation of single-tube agar-based test system. The principle of the method is that the enhancement of motility by TSB (0.4% [wt/vol] agar) medium and subsequent immunocapture of H7 flagellated bacteria in the middle agar layer containing H7 flagellar-specific antibodies result in retardation or stoppage of motility through the middle agar layer. FIG. 2 . Representative tubes showing motility of E. coli O157:H7 strains on MIO and TSBA media. Tube 1 indicates a motility score of 4 in MIO medium and also demonstrates the variegated or hazy appearance of this medium. Tubes 2, 3, and 4 show growth in TSBA medium with growth scores of 4, 3, and 2, respectively. FIG. 3 . Representative tubes showing reactions of E. coli O157:H7 with H7 antiserum adsorbed in the middle agar layer. (A) E. coli O157: H7 strain 36E1. Tubes 5 and 6 contain 30 μl of sterile saline solution (0.85% [wt/vol] NaCl) and 30 μl of H7 antiserum, respectively, per ml of TSBA medium. Tubes 7 and 8 contain 60 μl of saline solution and 60 μl of H7 antiserum, respectively, per ml of TSBA medium. Greater growth retardation is demonstrated in tube 8. (B) E. coli O157:H7 strain ATCC 43894. Tubes 9 and 10 contain 30 μl of sterile saline solution (0.85% [wt/vol] NaCl) and 30 μl of H7 antiserum, respectively, per ml of TSBA medium. Tubes 11 and 12 contain 60 μl of saline solution and 60 μl of H7 antiserum, respectively, per ml of TSBA medium. Greater growth retardation is demonstrated in tube 12.
Gram-negative bacteria possess at least six different mechanisms to actively transport proteins across the bacterial membranes (reviewed in reference 5). Of these six pathways, protein secretion induced upon contact of the bacteria with host cells has been referred to as the type III secretion pathway (10). Requirements of type III secretion pathways include the absence of a cleavable, hydrophobic amino-terminal signal sequence in the secreted protein, export of the protein across the bacterial inner and outer membranes without a periplasmic intermediate, and a signal to induce secretion (23). Most but apparently not all type III secreted proteins require chaperones (7, 8). We have demonstrated elsewhere that C. jejuni synthesize a novel set of proteins upon coculture with epithelial cells, some of which are secreted (22, 37). The secreted proteins were termed the Campylobacter invasion antigens (Cia proteins) because they were found to be required for maximal invasion of intestinal epithelial cells by C. jejuni (22, 30, 37). Because the Cia proteins are synthesized and secreted in response to an environmental stimulus and the secreted CiaB protein is not processed, the Cia proteins appear to conform to the criteria for type III proteins. However, a BLAST search of the C. jejuni genome revealed that the only apparent type III export system in C. jejuni is the flagellar apparatus.
A considerable amount of evidence exists that motility is essential for the maximal colonization of animals by C. jejuni (32, 33, 34, 36, 42). In parallel with these studies, additional work has been done to dissect the importance of motility versus the actual flagellum in the interaction of C. jejuni with cultured epithelial cells (15, 41, 43). Investigators have targeted genes encoding various flagellar structural components, and while discrepancies have been reported with respect to the phenotypes of particular mutants (14, 43), there appears to be a consensus among investigators that motility plays a role in C. jejuni pathogenesis. Moreover, motility and the expression of the flaA gene are clearly necessary for maximal invasion of eukaryotic cells and for the translocation of C. jejuni across polarized cells (15, 41). Perhaps more relevant to this study, differences in the invasive potential of C. jejuni flaA (flaB + ) and C. jejuni flaA flaB strains were noted in earlier studies C. jejuni flaA (flaB + ) strains have been reported to be more invasive than a C. jejuni flaA flaB strain (15, 41). Also noteworthy is that the invasiveness of a C. jejuni flaA (flaB + ) strain is enhanced 10-fold by promoting bacterium-host cell contact via centrifugation in contrast, the centrifugation step did not change the invasive potential of the C. jejuni wild-type strain (40). Based on the difference observed in the invasive potential of the C. jejuni flaA (flaB + ) strain versus the C. jejuni flaA flaB strain, Grant et al. (15) concluded that the flagellar structure played a role in internalization that was independent of motility. Prior to this study, it was unclear how the flagellum could have any effect on C. jejuni host cell invasion other than by conferring motility or acting directly as an adhesin.
Given our previous work suggesting that the Cia proteins are secreted in a type III-dependent manner and the absence of a type III secretion system dedicated to the export of virulence proteins in the C. jejuni genome, experiments were performed to determine if the flagellum serves as the Cia export apparatus. Mutations that abolished flagellin export (flhB, flgB, flgC, and flgE2), filament structure (fliD, flaA, flaB), and filament synthesis (fliA) were generated. With these mutants, we have shown that C. jejuni motility and virulence are linked. Specifically, we demonstrate that the C. jejuni Cia proteins are secreted via the flagellar export apparatus. The secretion system utilized by C. jejuni appears to be unique in that either one of the filament proteins is required for Cia protein secretion.
To test whether components of the flagellar apparatus serve as the C. jejuni Cia export apparatus, two separate experiments were performed. Mutations were generated in several flagellar structural genes in C. jejuni strain F38011 to determine if the loss of an operational flagellar apparatus resulted in the loss of Cia export. In addition, with two mutants of C. jejuni strain 81116 that were defective in expression of either one or both flagellin filaments as well as Cia protein export, we tested whether restoration of a flagellar filament also restored Cia protein synthesis. Mutations that affected either the export of flagellar components (flhB) or the nonfilament structural components (flgB, flgC, and flgE2) resulted in an S − phenotype. Comparable results were obtained with a second C. jejuni strain, 81116, in which the genes encoding the flagellin filament (flaA and flaB) were mutated. Complementation of the flagellar filament defect in 81116 with either flaA or flaB restored the organism's ability to secrete the Cia proteins. To ensure that the S − phenotype exhibited by the C. jejuni 81116 flaA flaB mutant was not unique to a particular strain, a C. jejuni F38011 flaA flaB mutant was generated. The C. jejuni F38011 flaA flaB mutant also exhibited an S − phenotype (not shown). Therefore, the genetic evidence presented is consistent with Cia protein secretion through the flagellar export system.
Insertion mutagenesis of fliD (Cj0548) and flgC (Cj0527c) was expected to have a polar effect on downstream gene expression. As the downstream genes in these putative operons are also expected to be associated with flagellar biosynthesis, we predicted that the phenotype associated with polarity on the downstream genes would be similar to that of the targeted gene. With regard to flgB and flgC, fliE (Cj0525c) and pbpB (Cj0524) would also be affected. In the case of fliD, fliS (Cj0549) and a hypothetical open reading frame of no known function (Cj0550) would have been affected.
The fact that the amount of FlaA protein was reduced in the whole-cell lysates of the C. jejuni flgB, flgC, and flgE2 mutants, as judged by immunoblot analysis with a flagellin antiserum, raised the possibility that C. jejuni may possess the FlgM anti-sigma factor. In bacteria such as Salmonella, Yersinia, and Helicobacter pylori, the negative regulator FlgM inhibits flagellin transcription in response to a defective hook-basal body complex (6, 9, 11). A protein corresponding to a putative FlgM homolog that shows similarity with the recently identified FlgM protein from H. pylori (9) has been identified in the genome of C. jejuni NCTC 11168 (Cj1464). In S. enterica serovar Typhi, σ 28 is involved in regulating gene expression of type III proteins (13). Given this fact, it is clear that the expression of virulence genes in S. enterica serovar Typhi is affected, albeit indirectly, by FlgM and the assembly of the flagellar export apparatus. Regardless, the Cia proteins are secreted in a C. jejuni fliA (σ 28 ) mutant. Therefore, the cia genes cannot be subject to transcriptional regulation via a mechanism involving the anti-sigma factor FlgM. Our results are in agreement with those of Jagannathan et al. (17), who observed that a C. jejuni fliA mutant displayed truncated flagella this finding indicates that σ 28 is not responsible for the transcription of the genes encoding the flagellar export apparatus in C. jejuni.
The ATPase FliI plays an essential role in flagellar apparatus assembly and in flagellar protein export. To address whether the S − phenotype of some of the C. jejuni mutants was due to regulatory effects, the expression of ciaB was analyzed in a C. jejuni F38011 fliI mutant. After the mutant was generated, RNA was extracted from bacteria that were cultured in Mueller-Hinton broth with 0.05% deoxycholate under microaerobic conditions. Importantly, ciaB was transcribed in the C. jejuni F38011 fliI mutant, as judged by reverse transcription-PCR. In addition, the CiaB protein was synthesized in the C. jejuni F38011 fliI mutant, as judged by immunoblot analysis with a CiaB-specific antibody.
The results of this study are consistent with the hypothesis that the flagellar type III secretion pathway is required for Cia protein export. Secretion of the Cia proteins requires a functional basal body and hook and at least one of the filament proteins. Coupled with the metabolic labeling experiments in which the C. jejuni strains were examined for protein secretion, the adherence and internalization data indicate that the difference in the invasiveness of the C. jejuni flaA flaB + and C. jejuni flaA flaB strains is a result of Cia secretion. Based on the phenotypes of the C. jejuni ciaB mutant (Mot + , S − ), it is also evident that motility, in the absence of Cia protein secretion, is not sufficient for C. jejuni invasion of epithelial cells. We believe that the data presented here reveal what had formerly been unclear about the Cia protein export apparatus and the relationship between C. jejuni motility and host cell invasion.
Identification of Motility and Autoagglutination Campylobacter jejuni Mutants by Random Transposon MutagenesisFIG. 1 . Location of transposon insertion sites within the C. jejuni genome. The nucleotide sequence flanking the insertion sites of 28 transformants was determined, and the insertion site positions within the C. jejuni NCTC11168 chromosome (36) were mapped. Transformants implicated in chemotaxis are indicated by bold type, while transformants implicated in AAG are indicated by underlining. FIG. 2 . Western blot analysis of surface FlaA from 28 motility mutants. C. jejuni outer membrane proteins were extracted with glycine-HCl and electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer of the gel, immunodetection was carried out with a polyclonal anti-FlaA antibody. Motility (Mot) is indicated as follows: +, 67 to 100% control motility ±, 31 to 66% control motility −, 0 to 32% control motility. AAG was determined in MH broth at RT for 3 h and is indicated as follows: +, AAG ±, altered AAG kinetics −, no AAG. flhA a is a defined flhA mutant of C. jejuni strain 81-176 (27), used as a negative control flhA b is a putative 480 flhA insertion mutant. FIG. 3 . Kinetics of AAG for 28 motility mutants. C. jejuni motility mutants were harvested following overnight growth and suspended in MH broth at RT. A 100-μl sample of the suspension was carefully removed at the time points indicated, and the OD600 was determined. All 28 mutants were analyzed however, lines were drawn for selected mutants for clarity. Cj1318, Cj1333, Cj1340c, and Cj1062 represent AAG mutants. A defined flhA mutant (0% motility and FlaA − ) (Fig. 2) was used as a negative control (27). Strains 480 and 81-176 were used as positive controls. FIG. 4 . Invasion assay of AAG mutants. Invasion is indicated as a percentage of control invasion. Strain 480 was used as a positive control, and flaA and flhA strains were used as negative controls. Error bars indicate the standard error for three experiments. FIG. 5 . AAG of C. jejuni on HeLa cells. Wild-type strain 480 (control) containing gfp and an isogenic flhA motility mutant containing gfp were allowed to adhere to HeLa cells for 1.5 h and were observed microscopically. (A and B) Strain 480 under phase-contrast and fluorescent conditions, respectively. (C and D) flhA mutant under phase-contrast and fluorescent conditions, respectively. FIG. 6 . C. jejuni NCTC11168 Cj1318 family. Shown is a C. jejuni NCTC11168 segment of DNA representing three of the four AAG mutants (Cj1318, Cj1333, and Cj1340c) as well as other Cj1318 family members, indicated in gray. neuC2, neuB2, neuB3, ptmA, and ptmB are known to affect the addition of sialic acid to C. jejuni proteins (12, 23). flaA and flaB are the genes for the major and minor flagellin subunits, respectively.
Isolation and identification of iron-reducing bacteria from gley soils
Seventy-one facultative anaerobic bacteria, capable of reducing iron oxide in pure culture, were isolated from three differently gleyed subsoils. The bacteria were picked at random from poured plates (10 −5 and 10 −6 ) inoculated with serially diluted soil samples. An attempt was made to identify these strains by morphological and biochemical tests. Among these 71 iron-reducing bacteria, all except three were capable of reducing nitrate to nitrite and 35 reduced nitrite further into gaseous compounds (denitrification), but only one strain (Bacillus subtilis) produced H2S. Based upon their physiological and morphological properties, 38 strains were allotted to the genus Pseudomonas, 31 sporeformers to the genus Bacillus and two were regarded to be coryneform (Arthrobacter?) bacteria. Species identified were Ps. denitrificans (23), ps. stutzeri (8) ps. fluorescens-putida (5), Bacillus cereus (6), B. cereus var. mycoides (14) and Bacillus subtilis (9). Two spore-forming bacilli, two non-pigmented pseudomonads and two coryneform type of bacteria could not be identified. The significance of the enzyme nitrate reductase (nitratase) of these bacteria for anaerobic respiration and as a mechanism of iron reduction is discussed.
Present address: Institut für Mikrobiologie der Technischen Hochschule, 61 Darmstadt, Rossdörferstrasse 140, Germany.
MATERIALS AND METHODS
Bacterial Strains and Plasmids.
Strains and plasmids used in this study are listed in Table Table1. 1 . pPA56 encodes the cytoplasmic signaling domain of Tsr (amino acids 290) (22). pMS103 encodes a similar domain of Tar (amino acids 257) fused to a leucine zipper at its N terminus in pMS105, amino acids 212 of Tar are also fused to a leucine zipper, but with an additional flexible linker region in between. The four sites of deamidation/methylation in Tar (QEQE) are mutated to produce QQQQ in these two plasmids (23). All three cytoplasmic fragments are reported to activate CheA kinase and confer a CW rotational bias to the flagella (22, 23).
Identification of a new chemoreceptor, Mcp4
A screen was performed to identify new chemoreceptors, or methyl-accepting chemotaxis proteins (MCPs), that may sense signals to direct motility in M. xanthus. Because these studies began before the availability of the M. xanthus genome sequence, we used a hybridization approach to identify new chemoreceptor genes. We took advantage of the highly conserved C-terminal domain (HCD) of MCP receptors, which is involved in transducing signals to downstream Che proteins ( Le Moual and Koshland, 1996 Zhulin, 2001 ). By aligning the sequences of MCPs from a variety of bacteria and archaea, a domain extending from amino acids 180 to 381 of DifA, the M. xanthus Dif system MCP, was identified as the highly conserved domain (Fig. 1B). A probe corresponding to the sequence encoding this domain was used to screen a cosmid library of the M. xanthus genome ( Hager et al., 2001 ). Cosmids that hybridized with the probe were analysed further by Southern blot analysis, and the fragments of interest were subcloned and sequenced. Of the six hybridizing cosmids, five contained the difA gene, and the remaining cosmid contained a previously unknown MCP homologue, which we termed mcp4. Mcp4 has two predicted transmembrane domains in the N-terminus as well as a HAMP domain (found in h istidine kinases, a denylyl cyclases, m ethyl-accepting proteins and p hosphatases), the HCD and four putative methylation sites in the C-terminus, suggesting that the receptor architecture and topology are similar to the E. coli Tar receptor (Fig. 1A). Mcp4 was used in a blast search ( Altschul et al., 1997 ) of the non-redundant protein database and was found to be most similar to Mcp3 from Rhodospirillum centenum, with 37% identity (E = 2 × 10 −39 ) over 334 amino acids of the conserved C-terminal domain. The N-terminal domain did not show similarity to any proteins in the database.
Mcp4 is homologous to known methyl-accepting chemotaxis proteins. A. Predicted domain structure of Mcp4 based on similarity to Tar, the E. coli aspartate receptor. Transmembrane regions are indicated by TM, thereby defining the periplasmic sensing domain. HAMP domains are found in many MCP homologues ( Zhulin, 2001 ), and predicted methylation sites are designated MH1 and MH2. The highly conserved domain (HCD) is present in all MCPs from bacteria and archaea. B. clustalw sequence alignment of the HCD of E. coli Tar and M. xanthus FrzCD, DifA and Mcp4. Dark boxes indicate sequence identity, and light shading indicates similarity.
Mcp4 is part of a chemotaxis-like gene cluster
By further sequencing of the region surrounding mcp4, we identified a fourth chemotaxis-like gene cluster on the M. xanthus genome. These sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number AY101609. This gene cluster was designated the che4 operon and is the focus of this paper (Fig. 2A). In addition to Mcp4, the che4 operon encodes two CheW homologues (CheW4A and CheW4B), a hybrid CheA–CheY (CheA4), a response regulator (CheY4) and a CheR homologue (CheR4). blastp searches with CheW4A and CheW4B indicate that these proteins are most similar to a chemotaxis signal transduction protein from Pseudomonas fluorescens PfO-1 with 35% identity over 74 amino acids (E = 4 × 10 −5 ) and 34% identity over 107 amino acids (E = 1 × 10 −10 ) respectively. CheR4 is 34% identical over 245 amino acids to the methyltransferase from E. coliO157:H7 EDL933 (E = 5 × 10 −27 ). In addition to the methyltransferase domain, CheR4 contains a 225-amino-acid C-terminal region containing a single tetratricopeptide repeat (TPR) domain, similar to that observed in FrzF and other CheR homologues. The function(s) of these domains in methyltransferases are unknown ( Shiomi et al., 2002 ). CheY4 is 38% identical over 122 amino acids to the receiver domain of a two-component response regulator from Nostoc sp. PCC 7120 (E = 4 × 10 −16 ), and CheA4 is 27% identical over 847 amino acids to a chemotaxis histidine protein kinase from Campylobacter jejuni (E = 8 × 10 −76 ). CheA4, like the kinases found in the M. xanthus Frz and Che3 pathways, also contains a C-terminal receiver domain. Although there are numerous hybrid CheA homologues in the GenBank database, the function of their receiver domains remains largely unknown. Notably, the che4 locus does not contain a gene encoding a CheB methylesterase homologue or a CheZ homologue. By analysing sequence downstream of cheA4, we identified a divergent open reading frame (ORF) with no homology to proteins in the blast database. Upstream (245 bp) of cheW4A, we identified a divergently transcribed gene that encodes a protein with 42% identity over 284 amino acids (E = 4 × 10 −55 ) to the kinase domain of a serine/threonine protein kinase from Thermoanaerobacter tengcongensis.
The che4 cluster comprises an operon. A. Genetic organization of the che4 region. Genes were named based on homology to known chemotaxis genes. The lines beneath the genes represent the regions that were PCR amplified in (B). B. DZ2 mRNA was reverse transcribed, and the resulting cDNA was detected by PCR amplification of regions A, B, C, or D as designated in (A). Product was detected on a 1% agarose gel, indicating that the genes in the locus are on the same transcript. (+) with reverse transcriptase (–) without reverse transcriptase.
The che4 gene cluster is transcribed as an operon
In order to determine whether the genes in the che4 locus are co-transcribed, reverse transcription polymerase chain reaction (RT-PCR) was performed on total RNA. Four different primer pairs were used to amplify regions from the cDNA spanning all the genes in the cluster (Fig. 2A). Primer set ‘A’ linked cheW4A and cheR4 primer set ‘B’ linked cheR4 and mcp4 primer set ‘C’ linked mcp4 and cheY4 and primer set ‘D’ linked cheY4, cheW4B and cheA4. Product was obtained for all four reactions, indicating that the genes in the che4 cluster are co-transcribed and, thus, form an operon (Fig. 2B). The absence of a product in the lanes lacking reverse transcriptase indicates that the signals were not the result of DNA contamination in the RNA preparation.
Isolation and analysis of mutants in the che4 operon
To determine whether the Che4 system functions to regulate motility, a deletion of the entire che4 operon (Δche4op) was constructed in the wild-type (A + S + ) strain, DZ2. Motility was measured by swarm diameter on nutrient-rich soft agar (0.3%), which favours S motility, and hard agar (1.5%), which allows cells to move using both A and S motility systems ( Shi and Zusman, 1993 ). The Δche4op mutant cells did not display observable motility defects under any of the conditions tested (Fig. 3A data not shown). However, M. xanthus can be propelled independently by either the A or the S motility system. It is possible that a subtle defect in the Δche4op mutant is masked in a wild-type strain that possesses both motility systems. To determine whether the Che4 system is involved in A or S motility, the che4 operon deletion was introduced into strains containing only S motility (A – S + ) or A motility (A + S – ). In an A + S – (pilA) strain, the Δche4op mutation appeared to be identical to the A + S – parent with regard to swarm size on hard or soft agar surfaces (data not shown) and morphology of the swarm edges (Fig. 3B), indicating that the che4 mutations had no effect on A motility. In contrast, in the A – S + (aglB1) strain, the Δche4op mutant showed enhanced swarming on hard agar surfaces. Furthermore, a deletion of the cheY4 gene caused a similar enhanced swarming phenotype in the A – S + background strain, indicating that CheY4 is the major output of the system (Fig. 4A). Surprisingly, no aberrant phenotype was observed on a soft agar (0.3%) surface in any of the mutants (data not shown).
The Δche4op mutation does not affect A motility. Cells were plated on rich media (CYE) with 1.5% agar and incubated at 32°C. A. Swarming of the A + S + strains. Pictures were taken after 2 days. B. Swarm edges are shown for the A + S – strains to demonstrate the single A + cells. Pictures were taken after 4 days.
The che4 mutants display S motility defects in the aglB1 background (A – S + ). A. Mutants containing deletions of the entire che4 operon, cheY4 and mcp4 in the A – (aglB1) background. Cells were plated on rich media (CYE) containing 1.5% agar and photographed after 4 days. All photos were taken at the same magnification. B. Colony edge of the aglB1Δche4op mutant. Pictures of the colony edge were taken after 5 days growth on rich media. C. The difA mutation is epistatic over the che4op deletion. Cells were plated on rich media plates containing 1.5% agar and photographed after 4 days.
Because the Δche4op and ΔcheY4 mutants only showed enhanced swarming in the A – S + parent and on hard agar surfaces, it was possible that deletion of the che4 operon restored A motility to the A – S + background. To test this possibility, we assayed the mutants for A motility by examining swarm edges. Figure 4B shows that the edges of the aglB1Δche4op mutant colonies were discrete and showed no evidence of single cells moving away from the colony edge, indicating that the Δche4op mutation did not restore A motility. Thus, the enhanced swarming displayed by the aglB1ΔcheY4 and aglB1Δche4op mutants must result from an effect on the S motility system. Together, these results suggest that the Che4 system acts as an inhibitor of S motility.
If the function of CheY4 is to inhibit S motility, then signalling through the chemoreceptor, Mcp4, should affect the inhibition. To test this hypothesis, a deletion of the mcp4 gene was constructed in the wild-type, A – and S – mutant backgrounds. As observed previously in the Δche4op and ΔcheY4 mutants, there was no difference in swarming patterns for the Δmcp4 mutant in the wild-type or A + S – strains (data not shown). The aglB1Δmcp4 strain did not show defects in motility on soft agar surfaces, similar to the ΔcheY4 and Δche4op mutants (data not shown). However, the aglB1Δmcp4 mutant had a reduced swarming phenotype on hard agar surfaces, which is the opposite of that seen in aglB1ΔcheY4 and aglB1Δche4op (Fig. 4A). These data indicate that Mcp4 signalling functions to alleviate inhibition of S motility mediated by CheY4.
Che4 mutants display wild-type levels of the S motility-associated structures
S motility requires the production and extrusion of type IV pili ( Kaiser and Crosby, 1983 Sun et al., 2000 ), extracellular matrix material (fibrils) ( Arnold and Shimkets, 1988 ) and LPS O-antigen ( Bowden and Kaplan, 1998 ). The aglB1ΔcheY4 and the aglB1Δche4op mutants, which showed enhanced swarming, and the aglB1Δmcp4 mutant, which showed reduced swarming, were analysed to determine whether they presented altered levels of these components. Similar levels of pilin were detected in all the strains by immunoblot analysis of whole-cell extracts using antibodies against PilA, the pilin monomer (data not shown). Furthermore, immunoblot analysis of pilin sheared off the cell surface showed no difference between the che4 mutants and the parent strain (Fig. 5A). The absence of pilin in the ΔpilQ strain (Fig. 5A, lane 5) ensures that preparations were not contaminated with intracellular pilin because PilQ is the secretin that is essential for pilin export. These results suggest that pilus assembly was unaffected in the mutants. Similarly, immunoblot analysis using antibodies specific for either FibA (a fibril-associated protein) or LPS O-antigen did not show altered levels of these cell surface components relative to the aglB1 parent (Fig. 5B and C). Extract from a difA mutant was included as a negative control for extracellular matrix material and FibA production. Consistent with the immunoblot analysis, Calcofluor white and Congo red dyes that stain the fibrils were able to bind the mutant cells in similar amounts to the parent strain (data not shown). Thus, all known structures required for S motility are present in the che4 mutants.
S motility components are present in the che4 mutants. Lanes 1 aglB1 lanes 2 aglB1Δmcp4 lanes 3 aglB1ΔcheY4 and lanes 4 aglB1Δche4op. For (A), lane 5, ΔpilQ lane 6, ΔpilA. For (B), lane 5, ΔpilA lane 6, difA. A. Surface pilin. Immunoblot of pili prepared by shearing probed with anti-PilA antiserum. B. Fibrils. Immunoblot of total protein isolated from cells grown to equal densities in liquid CYE medium probed with anti-FibA (mAb2105) antibody. C. LPS O-antigen. Whole-cell samples were prepared as in (B), but probed with anti-LPS O-antigen (mAb783) antibody.
The Dif system is required for Che4 function
The Dif system of M. xanthus contains homologues to chemotaxis genes and is required for extracellular matrix (fibril) production ( Yang et al., 1998 ). Extracellular matrix material is required for pilus retraction, and dif mutants are consequently hyperpiliated and defective in S motility ( Li et al., 2003 ). Because a deletion of the che4 operon results in cells that have enhanced S motility spreading (Fig. 4A), we wondered whether signalling through the Dif pathway was required for this enhancement. To address this possibility, triple mutants were generated in which the gene for difA (the dif system receptor) was disrupted in an aglB1Δche4op strain. Hard agar swarming assays of aglB1Δche4opdifA mutants were indistinguishable from the aglB1 difA parent, indicating that a difA mutant is epistatic to the Δche4op mutation (Fig. 4C). Similar analysis of the aglB1Δmcp4 difA and the aglB1ΔcheY4 difA mutants indicated that difA was epistatic to mcp4 and cheY4 (data not shown). These results allow us to conclude that the Che4 system requires either fibril biogenesis or functional type IV pili for the enhancement of S motility observed in the Δche4op and ΔcheY4 mutants.
Che4 mutants are defective in regulating cellular reversal frequencies
In order to understand better the effects of the various che4 mutations on motility, video microscopy was used to analyse single cell behaviour. We could not analyse cell movements using traditional techniques because the aglB1 strain lacks the ability to move as single cells on an agar surface. However, Sun et al. (2000 ) have demonstrated previously that single cells using only S-motility are able to move on a solid surface overlain with 1% methyl cellulose. Using this assay, we analysed the movement of cells with the following genotypes: aglB1, aglB1Δmcp4 and aglB1ΔcheY4. At least 50 individual cells were tracked for 30 min, and images were captured at 30 s intervals. Cell velocities and reversal frequencies were quantified for each strain, and a summary of the results is shown in Table 1. Linear regression analysis identified a strong inverse correlation between reversal frequency and velocity for aglB1 cells (Fig. 6A). For example, the slowest quartile of cells reversed on average once every 2.8 min, compared with the fastest quartile of cells, which reversed only once every 4.5 min. This suggests that there is a mechanism to co-ordinate reversal frequency and velocity. The correlation between velocity and reversal frequency was lost in aglB1Δmcp4 and aglB1ΔcheY4 mutants (Fig. 6A). Figure 6B summarizes the data observed in Fig. 6A with individual data points omitted for clarity. The increase in aglB1Δmcp4 reversal frequency (0.34 rev min −1 ) relative to aglB1ΔcheY4 (0.25 rev min −1 ) is statistically significant using Student's t-test (P < 1 × 10 −6 ). For comparison, when reversal frequency and velocity are plotted on an inverse graph (velocity on the y-axis), the average velocity of both mutants is almost the same (Fig. 6C), consistent with the values in Table 1.
|Average velocity (µm min −1 ) ± SEM||6.77 ± 0.43||6.11 ± 0.36||6.31 ± 0.40|
|Average reversal frequency (min −1 ) ± SEM||0.28 ± 0.01||0.25 ± 0.01||0.34 ± 0.01|
|Av. rev. for velocity < 6.00 µm min −1 ± SEM (no. of cells)||0.32 ± 0.01 (33)||0.26 ± 0.02 (35) a||0.33 ± 0.02 (27)|
|Av. rev. for velocity > 6.00 µm min −1 ± SEM (no. of cells)||0.25 ± 0.01 (33)||0.23 ± 0.02 (32)||0.34 ± 0.01 (23) a|
|% of cells exhibiting active movement (no. of cells evaluated)||82.5 (80)||87 (77)||86.2 (58)|
- Single cell motility was analysed, and the average values for cells exhibiting active movement are presented. SEM is the standard error of the mean.
- a. Values are significantly different from the other strains with a 95% confidence.
Reversal frequency correlates with velocity of aglB1 cells, but not of che4 mutants. A. Reversal frequency was compared with velocity using linear regression analysis, and P-values for the slope of each data set were obtained using a two-tailed t distribution. Each point indicates data for one cell analysed over a 30 min period. The number of cells analysed for each strain is as follows: aglB1, n = 66 aglB1ΔcheY4, n = 67 and aglB1Δmcp4, n = 50. B. Reversal frequency as a function of velocity. The trend lines obtained in (A) were overlain to demonstrate the difference in reversal frequency between the mutants and parent strains. Individual cell data points were omitted for clarity. C. Velocity as a function of reversal frequency. The data from (A) were plotted on opposite axes and overlain to compare the average velocities of the aglB1ΔcheY4 and aglB1Δmcp4 mutants. Individual cell data points were omitted for clarity.
The average reversal frequency for cells moving at different velocities cannot be compared because reversal frequency is dependent on velocity for the aglB1 strain. Therefore, in order to compare average reversal frequency between the che4 mutants and the parent strain, the data were divided into groups of cells moving more than or less than 6.0 µm min −1 (the median velocity for all the strains), and average reversal frequencies were obtained for these two populations (Table 1). The distribution of reversal rates was compared to determine whether they were statistically distinct at the 95% confidence level using an analysis of variance test followed by Tukey's multiple comparison test. This analysis showed that aglB1 cells moving with velocities < 6.0 µm min −1 reversed once every 3.1 min (0.32 rev min −1 ), significantly less than the 4.1 min between reversals (0.25 rev min −1 ) observed for cells moving with velocities > 6.0 µm min −1 . The reversal frequencies of cells moving more than or less than 6 µm min −1 were statistically indistinguishable for aglB1ΔcheY4 or aglB1Δmcp4 cells. At velocities < 6.0 µm min −1 , aglB1ΔcheY4 mutants reversed once every 3.8 min (0.26 rev min −1 ), which was significantly more than one reversal every 3.1 min (0.32 rev min −1 ) for aglB1. On the other hand, at velocities > 6.0 µm min −1 , aglB1Δmcp4 mutants reversed significantly less than aglB1 cells with frequencies of once every 2.9 min (0.34 rev min −1 ) and 4.1 min (0.25 rev min −1 ) respectively. aglB1ΔcheY4 reversed less frequently than aglB1Δmcp4 regardless of velocity.
The che4 operon is required for fruiting body formation in A – S + cells
Because motility is required for cells to aggregate and form fruiting bodies, we assayed expression of the che4 operon under developmental conditions. To follow expression of the che4 operon, an mcp4::lacZ gene fusion was constructed in the wild-type (A + S + ) background. β-Galactosidase activity was assayed in samples harvested at different times during development on CF starvation agar ( Kroos et al., 1986 ). Figure 7 shows that mcp4::lacZ was expressed under vegetative conditions however, there was as much as a 10-fold increase in expression by 48 h of development. These results suggest that the Che4 pathway also plays a role during fruiting body formation.
che4 expression increases during development. β-Galactosidase specific activity resulting from a mcp4::lacZ fusion was analysed at various time points after the onset of starvation. Zero hours of development indicates the time at which cells were shifted from rich media to starvation media. The graph represents the average of three experiments, and error bars depict standard error of the mean.
To determine whether the Che4 pathway was required for development, we analysed che4 mutants under starvation conditions. A – S + (aglB1) cells aggregated poorly on CF agar, but formed distinct fruiting bodies in submerged culture (Fig. 8A). When starved in the submerged culture assay, aglB1ΔcheY4 and aglB1Δche4op mutants did not aggregate at all. In contrast, the aglB1Δmcp4 mutant did aggregate (Fig. 8A). When heat- and sonication-resistant spores were quantified, the average number of spores obtained from aglB1 in submerged culture was 1.31 × 10 3 spores, compared with the DZ2 wild-type strain, which produced an average of 5.69 × 10 8 spores. The aglB1ΔcheY4 and the aglB1Δche4op mutants displayed at least a fivefold reduction in levels of spores relative to the aglB1 parent (five and 30 spores respectively). Conversely, the aglB1Δmcp4 mutant produced nine times the level of spores of the aglB1 parent (Fig. 8B). Despite the increase in sporulation in aglB1Δmcp4 relative to aglB1, the level of sporulation is still 10 4 -fold lower then the wild-type A + S + strain. It is worth noting that, although the aglB1ΔcheY4 and the aglB1Δche4op mutants were unable to aggregate, they formed a cohesive film in the submerged culture assay, indicating that pili and other factors responsible for cell–cell contacts were functional. Thus, the effects on developmental aggregation and sporulation seen in various che4 mutants are attributable to an inability to regulate S motility rather than to changes in the S motility apparatus.
Development is affected by mutations in the che4 operon. A. The aglB1ΔcheY4 and aglB1Δche4op mutants do not aggregate when starved. Cells were grown in submerged culture in MMC buffer for 7 days before photographing. B. Viable spores were measured by plating 7-day-starved cells after heat and sonication treatment. The data are the average of at least three experiments, and the number of spores was normalized to the aglB1 parent. Error bars depict the standard error of the mean. The percentage of spores relative to the aglB1 parent is shown above the bars.
Fig.S1. Expression profiles of motility genes in broth, on 0.6% plates or on 1.5% plates.
TableS1. Original time course microarray data for S. typhimurium in LB broth or on 0.6% or 1.5% LB plates.
TableS2. Full gene list of eight differential expression groups of S. typhimurium under three growth conditions (LB broth, 0.6% or 1.5% LB plates).
Table S1. Original time course microarray data for S. typhimurium in LB broth or on 0.6% or 1.5% LB plates.
Table S2. Full gene list of eight differential expression groups of S. typhimurium under three growth conditions (LB broth, 0.6% or 1.5% LB plates).
Fig. S1. Expression profiles of motility genes in broth, on 0.6% plates or on 1.5% plates.
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