Biofilms are aggregates of bacterial cells enclosed within a self-generated extracellular matrix, forming a three-dimensional structure that allows the bacteria cells to firmly attach to both living and non-living surfaces. The extracellular matrix of Salmonella enterica serovar Typhimurium (STm) biofilms is composed of curli, cellulose, BapA, O-antigen capsule, colanic acid and extracellular DNA (eDNA) and protects the resident bacteria from diverse environmental stresses such as antibiotics and the host immune response. Studies have shown that curli, which accounts for 85% of the extracellular matrix, plays a significant role in the establishment of the matrix. Curli interacts with eDNA and cellulose to create a mesh-like network that stabilizes the biofilm, aids in surface adherence, and confers elasticity to the biofilm. Although curli, cellulose, eDNA, and their interactions have been studied, it is not clear how BapA, O-antigen capsule, and colanic acid contribute to Salmonella biofilm structure. Here, we examined the individual role of the matrix components utilizing various isogenic mutants for curli (csgBA), cellulose (bcsE, bcsA), enterobacterial common antigen (wecA), BapA protein (bapA), colanic acid (wza-wcaM), O-antigen capsule (yihQ) and csgD, a master regulator for biofilm formation. We characterized the colony biofilm morphology by Congo red-Coomassie blue staining and the biomass in pellicle biofilms by crystal violet assay. Next, we utilized a computational 4D approach that was developed in collaboration with the Quiesser and Buttaro Labs to identify the contribution of each matrix component into the material properties of the biofilm. Finally, by utilizing the above-mentioned assays, we explored the influence of different culture media on biofilm formation and architecture comparing Tryptic Soy broth (TSB) and No salt Luria Broth (LB No salt) media for biofilm growth. In our studies, we found that only curli-containing strains maintained the ability to form biofilms comparable to wildtype STm and irrespective of the media condition, indicating a key role for curli in the establishment of biofilms. However, when characterizing the physical properties of the biofilms utilizing the 4D assay, we saw a different phenotype between the two culture media conditions. We observed that when biofilms were formed in LB No salt culture condition, curli was the most significant contributor to the rigidity of biofilms such that, in the absence of curli, biofilms were more fluid-like and less rigid when compared to curli-containing strains. In TSB media, curli production was decreased although there was no loss of biofilm rigidity. Overall, our data suggests that different culture conditions could lead to different material properties of STm biofilm matrix, which in turn affects the physical properties such as the rigidity of the biofilms. Additionally, the biofilm matrix components may play redundant roles and another component could compensate for the loss of curli. / Biomedical Sciences

Maize (Zea mays L) is a major crop plant belonging to the family Poaceae, which includes other important cereals such as wheat, rice, barley and oats (Kellogg, 1998). Other grass crops are rye, common millet, finger millet, teff, sugarcane and sorghum. Many of these are important food crops in most parts of the world, including Africa. The sustainable production of sufficient and affordable food for a rapidly increasing population is one of Africa's greatest challenges.

The solventogenic Clostridium acetobutylicum strain P262 was used for the commercial production of solvents in South Africa from 1945 to 1983 (Jones and Woods, 1986a). Our laboratory has focused on understanding two: fundamental aspects of its physiology, namely nitrogen metabolism and electron transport pathways. The long-term goal is the potential genetic modification of nitrogen utilization and/or electron distribution in the cell, to manipulate fermentation patterns for improved growth rate and solvent yields. The main aims of this research project were to: extend the sequence analysis of the glnA locus encoding the glutamine synthetase (GS) enzyme (Janssen et al., 1990; Fierro-Monti et al., 1992); to identify and characterize the locus encoding the structural genes for the glutamate synthase (GOGAT) enzyme; and to detennine the mechanisms and growth conditions which regulate the activity of these two key enzymes of nitrogen assimilation. In addition, we were interested in characterizing clone pMET13Cl, which was isolated by a selection system developed to clone genes involved in electron transport from C. acetobutylicum (Santangelo et al., 1991). Sequence analyses revealed that the region downstream of glnA and the putative regulatory gene, glnR (Reid and Woods, 1995), encoded the structural genes for the large (a) and small (J) subunits of GOGAT, respectively. This is the first report in which the genes encoding GS and GOGAT are genetically linked. The a subunit was designated gltA, and the downstream f3 subllll.it was designated gltB. All the likely cofactor and substrate binding sites identified in GOGAT enzymes (Vanoni and Curti, 1999) were conserved in the deduced gltA and gltB polypeptides. The identity of the gltA and gltB genes were functionally confirmed by complementation studies involving their co-expression from separate constructs in an E. coli glutamate auxotroph (strain: Mx:3004), which restored the ability of this mutant to grow with ammonia as the sole source of nitrogen. Physiological studies on the germination, growth and differentiation patterns of C. acetobutylicum P262 were assessed in relation to different nitrogen conditions. Significantly, organic nitrogen (casamino acids) was the preferred source of nitrogen, and not ammonia as ll previously asswned. These studies led to the development of nitrogen limiting conditions (0.025% casamino acids + 0.15% glutamine) and nitrogen rich conditions (0.2% casamino acids) used for regulatory studies. GOG AT activity was optimised. It appeared to be sensitive to oxygen and specific for the co-enzyme NADH. Both GS and GOGAT activities were regulated by the nitrogen source in a similar way: induced in the nitrogen limiting conditions and repressed in the nitrogen rich conditions. Northern blot analyses, in conjunction with the enzyme activity profiles and feedback inhibition studies, indicated that GS and GOGAT activities were regulated primarily at the level of transcription. Furthermore, glnA. andglnR, and gltA and gltB are each transcribed as an operon under nitrogen limiting conditions. No assimilatory GDH activity could be detected. The implications of these results, as well as sequence features identified, are discussed in context with a proposed model for the regulation of GS and GOGAT activity in C. acetobutylicum P262. Analysis of plasmid pMET13Cl identified a gene whose predicted -46 kDa product was associated with an electron transport function. The deduced amino acid sequence was not typical of electron transport proteins, but rather shared striking homology to bacterial GOGAT p subunit polypeptides. This p subunit-like gene was thus designated gltX. We were, however, unable to relate it to GOGAT activity or nitrogen metabolism. Rather, it appears to belong to a novel family of FAD-dependent NAD(P)H oxidoreductases suggested by Vanoni and Curti (1999) and supported by an analysis of the evolutionary relationships of the GOGAT subunits/domains from various sources.

During the 1994-95 growing season, an apparently new disease was observed on Brassica oleracea var. capitata (cabbage) seedlings in nurseries of the province of KwaZulu-Natal, South Africa. Named chocolate spot disease of crucifers because of the characteristic symptoms of dark brown to black lesions with transparent centers, this disease was particularly severe on the more mature leaves of stressed seedlings and resulted in serious economic losses. The causal agent, determined to be a strain variant of Xanthomonas campestris pv. campestris was found to be associated with bacteriophage-like particles. The optimal growth conditions for production of the bacteriophage-like particles by the chocolate spot pathogen were determined and a purification protocol developed that yielded particles in a quantity and quality adequate for further analysis. Based on a comparison of the particle and bacterial protein reactions with the antiserum raised against the purified particles, it was concluded that the particles were not vesicles originating from the bacterial membrane, but were therefore most probably of bacteriophage origin. Physicochemical characterisation showed that the putative phage is a tailless isometric particle with a diameter of 33.45 nm, sedimentation coefficient of 85S and a density in CsCl of 1.347 g/ml. The phage is relatively stable with respect to pH, solvents and at temperatures of 40°C and 60°C. The particles contain 3 major proteins of 94, 32 and 21.8 kDa as well as two minor proteins of 40 and 25.7 kDa, and it has a single-stranded DNA genome. Biological characterisation of the novel bacteriophage indicates that it is a temperate phage which does not cause visible plaque formation on solid media and is not inducible by the external factors, mitomycin C and ultraviolet radiation. Serological tests showed that the phage is present in all isolates of the chocolate spot pathogen and that similar particles are associated with the X. campestris pathovars aberrans, armoraciae, campestris and raphani. No serological relationship was detected between the novel phage and the X. c. pv. campestris phage, RR68. However, the novel phage antibodies recognised 3 proteins of molecular weights 21.8, 13.79 and 12.64 kDa in the X. c. pv. campestris phage, HT3h. These proteins are localised in the phage capsid. Although phage-like particles were detected in the novel phage host PCB 22 following electron microscopy of ultra-thin sections, immunogoldlabelling could not confirm whether the particles were of phage origin. The single-stranded DNA genome of the phage hybridised with a 53.9 kb extrachromosomal DNA element. Since its size precludes this element being packaged as single-stranded DNA into a capsid 33.45 nm in diameter, it is most probable that this 53.9 kb DNA element is an indigenous plasmid into which the double-stranded form of the phage genome has integrated. However, this could not be confirmed by the results of the nucleic acid hybridisation tests. The 53.9 kb extrachromosomal element was cloned and several recombinant plasmids sequenced. No typical phage genes were identified. However, fragments of a Xanthomonas avirulence (avr) gene interrupted by part of a transposon sequence were identified. A 201- 262 base region of clone pSSI shared a 98% identity to the 3' end of a group of Xanthomonas avr genes, while 141-311 bases had a high degree of nucleotide similarity to the 5' end of the avr genes. These two regions of avr gene similarity which appear to converge, are interrupted by 1259 bases sharing 99% nucleotide identity to the 4864-6126 bp position of the 6938 bp X. campestris transposon, ISXC5 and 298 bases with 97% similarity to the 6644-6938 bp region of the transposon. The 1259 and 298 base regions are in tum separated by an unsequenced region of 518 bases. The presence of the avr and transposon sequences (usually located on plasmids or the bacterial chromosome) on the extrachromosomal element strengthens the hypothesis that the phage genome is integrated into an indigenous plasmid.

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