Flavobacterium johnsoniae PorV Is Required for Secretion of a Subset of Proteins Targeted to the Type IX Secretion System

Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.     

A novel locus essential for spreading of Cytophaga hutchinsonii colonies on agar

Cytophaga hutchinsonii is an aerobic cellulolytic gliding bacterium. The mechanism of its cell motility over surfaces without flagella and type IV pili is not known. In this study, mariner-based transposon mutagenesis was used to identify a new locus CHU_1797 essential for colony spreading on both hard and soft agar surfaces through gliding. CHU_1797 encodes a putative outer membrane protein of 348 amino acids with unknown function, and proteins which have high sequence similarity to CHU_1797 were widespread in the members of the phylum Bacteroidetes. The disruption of CHU_1797 suppressed spreading toward glucose on an agar surface, but had no significant effect on cellulose degradation for cells already in contact with cellulose. SEM observation showed that the mutant cells also regularly arranged on the surface of cellulose fiber similar with that of the wild type strain. These results indicated that the colony spreading ability on agar surfaces was not required for cellulose degradation by C. hutchinsonii. This was the first study focused on the relationship between cell motility and cellulose degradation of C. hutchinsonii.     

Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5′ region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.     

Flavobacterium johnsoniae gldN and gldO Are Partially Redundant Genes Required for Gliding Motility and Surface Localization of SprB

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Mutations in gldN cause a partial defect in gliding. A novel bacteriophage selection strategy was used to aid construction of a strain with a deletion spanning gldN and the closely related gene gldO in an otherwise wild-type F. johnsoniae UW101 background. Bacteriophage transduction was used to move a gldN mutation into F. johnsoniae UW101 to allow phenotypic comparison with the gldNO deletion mutant. Cells of the gldN mutant formed nonspreading colonies on agar but retained some ability to glide in wet mounts. In contrast, cells of the gldNO deletion mutant were completely nonmotile, indicating that cells require GldN, or the GldN-like protein GldO, to glide. Recent results suggest that Porphyromonas gingivalis PorN, which is similar in sequence to GldN, has a role in protein secretion across the outer membrane. Cells of the F. johnsoniae gldNO deletion mutant were defective in localization of the motility protein SprB to the cell surface, suggesting that GldN may be involved in secretion of components of the motility machinery. Cells of the gldNO deletion mutant were also deficient in chitin utilization and were resistant to infection by bacteriophages, phenotypes that may also be related to defects in protein secretion.Cells of Flavobacterium johnsoniae, and of many other members of the phylum Bacteroidetes, crawl over surfaces at approximately 2 μm/s in a process called gliding motility. F. johnsoniae cells glide on agar, glass, polystyrene, Teflon, and many other surfaces (16, 22). Cells suspended in liquid also bind and propel added particles such as polystyrene latex spheres (23). The mechanism of this form of cell movement is not well understood despite decades of research (15). Genome analyses suggest that F. johnsoniae gliding is genetically unrelated to other well-studied forms of bacterial movement such as bacterial flagellar motility, type IV pilus-mediated twitching motility, myxobacterial gliding motility, and mycoplasma gliding motility (10, 20, 21). Genes and proteins required for F. johnsoniae motility have been identified (1-3, 7-9, 17, 18). GldA, GldF, and GldG appear to form an ATP-binding cassette transporter that is required for gliding (1, 7). Eight other Gld proteins (GldB, GldD, GldH, GldI, GldJ, GldK, GldL, and GldM) are also required for movement (2, 3, 8, 9, 17, 18). Many of these are unique to members of the phylum Bacteroidetes. Disruption of the genes encoding any of these 11 proteins results in complete loss of motility. The mutants form nonspreading colonies, and individual cells exhibit no movement on agar, glass, Teflon, and other surfaces tested. The Gld proteins are associated with the cell envelope and presumably constitute the gliding motor, but none of them appear to be exposed on the cell surface. Mutations in sprA and sprB, which encode cell surface proteins, result in partial motility defects. Cells form nonspreading colonies, but some of the cells exhibit limited movement in wet mounts. SprA is required for efficient attachment to glass (22), and SprB appears to be a mobile adhesin that is propelled along the cell surface by the gliding motor and thus transmits the force generated by the motor to the surface over which cells crawl (10, 21). The surface localization of SprA and SprB and the phenotypes of sprA and sprB mutants suggest that the gliding motor is at least partially functional in these mutants but that force is inefficiently transmitted to the substratum. Analysis of the F. johnsoniae genome revealed the presence of multiple paralogs of sprB, which may explain the residual motility of sprB mutants (20).gldN lies downstream of gldL and gldM, and the three genes constitute an operon (2). Cells with transposon insertions in gldN form nonspreading colonies that are indistinguishable from those of other gld mutants. However, unlike other gld mutants, gldN mutants exhibit some residual ability to glide in wet mounts (2). One possible explanation for this phenotype is that GldN may have a peripheral and nonessential role in gliding. Alternatively, GldN may perform a critical function in gliding, but in its absence another cellular protein may compensate for the missing GldN function. F. johnsoniae has a gldN paralog, gldO, that is located downstream of gldN but is transcribed independently (2). The GldN and GldO proteins are 85% identical over their entire lengths, making GldO a prime candidate for a protein that might compensate for lack of GldN.Recent results suggest that some of the F. johnsoniae Gld and Spr proteins, including GldN, may be components of a novel bacteroidete protein translocation apparatus referred to as the Por secretion system (PorSS) (28). This conclusion emerged from studies of gingipain protease secretion by the distantly related nonmotile bacteroidete Porphyromonas gingivalis. P. gingivalis is a human periodontal pathogen, and gingipain proteases are important virulence factors. Gingipains have signal peptides that allow export across the cytoplasmic membrane via the Sec machinery, but they rely on components of the PorSS for secretion across the outer membrane (27-29). P. gingivalis cells with mutations in genes homologous to F. johnsoniae gldK, gldL, gldM, gldN, and sprA are defective in gingipain secretion across the outer membrane (28). F. johnsoniae has a homologue to another P. gingivalis gene required for gingipain secretion, porT. Disruption of the F. johnsoniae porT homologue (referred to as sprT) results in motility defects and defects in surface localization of SprB (28).This study was designed to identify possible roles for GldN in motility and to determine whether GldN and GldO are partially redundant components of the motility apparatus. The results demonstrate that F. johnsoniae GldN has an important function in motility and that GldO can replace GldN in this role. They suggest that GldN is needed for efficient secretion of the cell surface motility protein SprB, which may explain some of the motility defects of the gldN mutants.     

The volatile-producing Flavobacterium johnsoniae strain GSE09 shows biocontrol activity against Phytophthora capsici in pepper

Aims: Previously, we selected a bacterial strain (GSE09) antagonistic to Phytophthora capsici on pepper, which produced a volatile compound (2,4‐di‐tert‐butylphenol), inhibiting the pathogen. In this study, we identified strain GSE09 and characterized some of the biological traits of this strain in relation to its antagonistic properties against P. capsici. In addition, we examined bacterial colonization on the root surface or in rhizosphere soil and the effect of various concentrations of the volatile compound and strain GSE09 on pathogen development and radicle infection as well as radicle growth. Methods and Results: Strain GSE09 was identified as Flavobacterium johnsoniae, which forms biofilms and produces indolic compounds and biosurfactant but not hydrogen cyanide (HCN) with little or low levels of antifungal activity and swimming and swarming activities. Fl. johnsoniae GSE09 effectively colonized on pepper root, rhizosphere, and bulk (pot) soil, which reduced the pathogen colonization in the roots and disease severity in the plants. Various concentrations of 2,4‐di‐tert‐butylphenol or strain GSE09 inhibited pathogen development (mycelial growth, sporulation, and zoospore germination) in I‐plate (a plastic plate containing a center partition). In addition, germinated seeds treated with the compound (1–100 μg ml^?1) or the strain (10²–10¹⁰ cells ml^?1) significantly reduced radicle infection by P. capsici without radicle growth inhibition. Conclusions: These results indicate that colonization of pepper root and rhizosphere by the Fl. johnsoniae strain GSE09, which can form biofilms and produce indolic compounds, biosurfactant, and 2,4‐di‐tert‐butylphenol, might provide effective biocontrol activity against P. capsici. Significance and Impact of the Study: To our knowledge, this is the first study demonstrating that the Fl. johnsoniae strain GSE09, as a potential biocontrol agent, can effectively protect pepper plants against P. capsici infection by colonizing the roots.     

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6′‐phosphate phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose‐specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6‐phospho‐α‐glucosidase, which in B. subtilis hydrolyses maltose 6′‐P into glucose and glucose 6‐P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6‐P into glucose 1‐P and glucose 6‐P. However, purified MalP phosphorolyses maltose but not maltose 6′‐P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6′‐P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1‐P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6′‐P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6′‐P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1‐P. MapP therefore connects PTS‐mediated maltose uptake to maltose phosphorylase‐catalysed metabolism. Dephosphorylation assays with a wide variety of phospho‐substrates revealed that MapP preferably dephosphorylates disaccharides containing an O‐α‐glycosyl linkage.     

TheamrG1 gene is involved in the activation of acetate inCorynebacterium glutamicum

During growth ofCorynebacterium glutamicum on acetate as its carbon and energy source, the expression of theptaack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genesamrG1 andamrG2 found in the deregulated transposon mutant C.glutamicum G25. TheamrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C.glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain ofamrG1 in the C.glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, theamrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of theamrG1 gene in C.glutamicum 13032 had the adverse regulatory effect. These results indicate that theamrG1 gene encodes a repressor or co-repressor of theptaack operon.     

TheamrG1 gene is involved in the activation of acetate inCorynebacterium glutamicum

During growth ofCorynebacterium glutamicum on acetate as its carbon and energy source, the expression of theptaack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genesamrG1 andamrG2 found in the deregulated transposon mutant C.glutamicum G25. TheamrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C.glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain ofamrG1 in the C.glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, theamrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of theamrG1 gene in C.glutamicum 13032 had the adverse regulatory effect. These results indicate that theamrG1 gene encodes a repressor or co-repressor of theptaack operon.     

Functional specialization and differential regulation of short‐chain carboxylic acid transporters in the pathogen Candida albicans

The major fungal pathogen Candida albicans has the metabolic flexibility to assimilate a wide range of nutrients in its human host. Previous studies have suggested that C. albicans can encounter glucose‐poor microenvironments during infection and that the ability to use alternative non‐fermentable carbon sources contributes to its virulence. JEN1 encodes a monocarboxylate transporter in C. albicans and we show that its paralogue, JEN2, encodes a novel dicarboxylate plasma membrane transporter, subjected to glucose repression. A strain deleted in both genes lost the ability to transport lactic, malic and succinic acids by a mediated mechanism and it displayed a growth defect on these substrates. Although no significant morphogenetic or virulence defects were found in the double mutant strain, both JEN1 and JEN2 were strongly induced during infection. Jen1‐GFP (green fluorescent protein) and Jen2‐GFP were upregulated following the phagocytosis of C. albicans cells by neutrophils and macrophages, displaying similar behaviour to an Icl1‐GFP fusion. In the murine model of systemic candidiasis approximately 20–25% of C. albicans cells infecting the kidney expressed Jen1‐GFP and Jen2‐GFP. Our data suggest that Jen1 and Jen2 are expressed in glucose‐poor niches within the host, and that these short‐chain carboxylic acid transporters may be important in the early stages of infection.     

Mutation of rpoS enhances Pseudomonas sp. KL28 growth at higher concentrations of m-cresol and changes its surface-related phenotypes

A Tn5 transposon mutant was isolated of the alkylphenol degrader Pseudomonas sp. KL28 that showed increased growth at higher levels of m-cresol on solid and in liquid cultures. The transposon was inserted at the 5'-terminus of rpoS, which encodes a stationary-phase sigma factor. When grown on agar plates, the rpoS mutant developed prominent wrinkles, especially at lower temperatures, and spread faster on soft agar. In addition, the rpoS mutant had enhanced biofilm-forming ability that was not due to self-produced diffusible signals.     

Identification of the Flavobacterium johnsoniae cysteate-fatty acyl transferase required for capnine synthesis and for efficient gliding motility

Sulfonolipids (SLs) are bacterial lipids that are structurally related to sphingolipids. Synthesis of this group of lipids seems to be mainly restricted to Flavobacterium, Cytophaga and other members of the phylum Bacteroidetes. These lipids have a wide range of biological activities: they can induce multicellularity in choanoflagellates, act as von Willebrand factor receptor antagonists, inhibit DNA polymerase, or function as tumour suppressing agents. In Flavobacterium johnsoniae, their presence seems to be required for efficient gliding motility. Until now, no genes/enzymes involved in SL synthesis have been identified, which has been limiting for the study of some of the biological effects these lipids have. Here, we describe the identification of the cysteate-fatty acyl transferase Fjoh_2419 required for synthesis of the SL precursor capnine in F. johnsoniae. This enzyme belongs to the α-oxoamine synthase family similar to serine palmitoyl transferases, 2-amino-3-oxobutyrate coenzyme A ligase and 8-amino-7-oxononanoate synthases. Expression of the gene fjoh_2419 in Escherichia coli caused the formation of a capnine-derived molecule. Flavobacterium johnsoniae mutants deficient in fjoh_2419 lacked SLs and were more sensitive to many antibiotics. Mutant growth was not affected in liquid medium but the cells exhibited defects in gliding motility.     

Characterization of glucose transport inSchizosaccharomyces pombe

Conclusions GHT1 was isolated as suppressor ofd-glucose uptake deficiency ofS. pombe mutant YGS-5. The correspondingS. pombe DNA encodes a putative protein with significant amino acid sequence identity to theS. cerevisiae HXT transporters. Heterologous expression ofGHT1 inS. cerevisiae hxt mutant RE700A (strain HLY709) enabled the mutant to grow ond-glucose as the sole carbon source. HLY709 cells take up hexoses with similar specificity toS. pombe wild strain and accumulate the non-metabolizable analogues of glucose (2DG and 6DG) intracellularly, thus matchingS. pombe wild strain. Southern blot analysis revealed the existence of other putative glucose transporters inS. pombe and the search for related transporter genes inS. pombe genome is in progress.     

Novel Features of the Polysaccharide-Digesting Gliding Bacterium Flavobacterium johnsoniae as Revealed by Genome Sequence Analysis

The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the “motor” and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae.Flavobacterium johnsoniae (formerly Cytophaga johnsonae) is a member of the large and diverse phylum of gram-negative bacteria known as the Bacteroidetes. Members of this group of organisms have a number of unique characteristics that distinguish them from other bacteria. Some have novel cell surface machinery to utilize polysaccharides (85, 95, 96). Rapid gliding motility over surfaces is also common among these bacteria (59), as are unusual outer membrane sulfonolipids (29) and flexirubin pigments (78). Bacteroidete gene expression and regulation also have novel aspects (10, 11, 20, 39, 92). The many unusual features of these common but understudied bacteria provide numerous avenues for further exploration, which can be greatly aided by analysis of genome sequences.F. johnsoniae digests many polysaccharides and proteins, but it is best known for its ability to rapidly digest insoluble chitin (87). Chitin is one of the most abundant biopolymers on earth (63). F. johnsoniae and other members of the Bacteroidetes phylum are thought to play important roles in the turnover of this compound in many environments (47). F. johnsoniae has become a model system for the study of bacteroidete gliding motility biochemistry and molecular biology (20, 27-29, 59, 72). This paper highlights novel features of the F. johnsoniae genome, with particular emphasis on genes and proteins likely to be involved in polysaccharide utilization, gliding motility, and the novel biochemistry of this organism.     

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.     

The role of regulation of cell speed in the behavior of Physarum polycephalum amoebae

Studies on the behavior of wild-type and mutant Physarum polycephalum amoebae have revealed that regulation of cell speed results in different patterns of cell dispersion in different environments and have shown that P. polycephalum can be used for genetic studies of the mechanisms responsible for this element of cell behavior. Colonies generated by clonal populations of amoebae growing on E. coli display alternate colony morphologies depending on the pH of the culture medium and the presence of live E. coli as a nutrient. In the larger ‘spreading colonies’ cells at the outside of a colony are dispersed over a wide band of bacteria while in the smaller ‘aggregate ring colonies’ most cells moving on bacteria are aggregated in a regularly shaped ring on a narrow band of bacteria at the border of the bacterial lawn created when amoebae completely consume the bacteria available in the colony center. Measurements of cell growth, the rate of colony expansion, and the rate of single cell movement show that cells in contact with bacteria move more slowly in aggregate ring than in spreading colonies. Moreover, since in aggregate ring colonies the rate of movement of cells in contact with bacteria is also reduced relative to that of cells moving on adjacent regions of the agar surface, inhibition of cell speed appears to be at least partially responsible for generating the aggregate ring morphology. Characterization of the behavior of a single locus mutant which generates spreading colonies under conditions where aggregate ring colonies are normally formed has provided additional evidence that a specific mechanism is involved in controlling the distribution of amoebae through regulation of cell speed. Furthermore, the studies of this mutant have shown that aberrant colony morphology can be used as an easily recognized phenotype for identifying and studying mutants with defects which affect the regulation of cell speed.     

CtaA of Staphylococcus aureus Is Required for Starvation Survival, Recovery, and Cytochrome Biosynthesis

A Staphylococcus aureus mutant (SPW3) apparently unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous to ctaA of Bacillus subtilis which encodes a heme A synthase. Analysis of the cytochrome profiles of SPW3 revealed the absence of heme A-containing cytochromes compared to the parental 8325-4 strain. SPW3 demonstrated a 100-fold reduction in the ability to survive starvation induced by glucose limitation, under aerated conditions, compared to 8325-4. Analysis of starved cultures revealed that greater than 90% of the cells which demonstrated metabolism (as shown by rhodamine 123 accumulation) were unable to recover and form colonies on agar. Analysis of the lag phase and initial growth kinetics of those cells which could recover also showed a defect. This recovery defect could be partially alleviated by the inclusion of catalase in the recovery medium, indicating the probable involvement of oxidative stress. SPW3 also exhibited reduced colony size similar to that of a small-colony variant, increased resistance to aminoglycoside antibiotics, and reduced hemolysin and toxic shock syndrome toxin 1 production, but no alteration in the ability to form lesions in a subcutaneous mouse infection model.