Bacteria can actively communicate and interact with each other to establish multicellular communities. Many of these processes involve functional differentiation of cells into specialised subpopulations by expression of varying genetic programmes. This leads to division of labour between the arising subpopulations of cells in the community. One type of such community is the biofilm, which is composed of microbial cells enclosed in a biopolymeric matrix. Such biofilms can be formed in a large range of environments from sea beds to animal tissues. Bacillus subtilis is a soil dwelling Gram-positive rod that was shown to closely interact with plants and establish a protective symbiosis by formation of biofilms on the roots. The biofilm matrix synthesised by B. subtilis is composed of the exopolysaccharide, for which the chemical structure is not yet established, and a protein TasA that forms amyloid-like fibres spanning between the cells and anchored to the cells by an accessory protein TapA. A third protein of unknown function, YuaB, has also been shown to be necessary for establishment of a biofilm. However, the mechanism of function for YuaB has not been elucidated. The data presented in this report focus on the role played by YuaB during formation of the biofilm. By analysis of cell differentiation patterns YuaB was shown to be required for maturation of the biofilm. The localisation of YuaB is identified in two “subtypes” of biofilm, a biofilm pellicle floating on the air-liquid interface and complex colonies formed on solid surfaces. This is achieved using a combination of biofilm fractionation combined with Western blotting and a newly developed method for immuno-fluorescent labelling of biofilm proteins. YuaB acts in synergy with the exopolysaccharide and TasA, as both components of the biofilm matrix are synthesised in the absence of YuaB but the biofilm is not made. The initial structural characterisation of YuaB is presented based on in silico predictions and physiological and biophysical analysis of the mutations introduced into the sequence of YuaB. The experimental data is concluded with a hypothesis that YuaB forms a hydrophobic protective layer necessary for support of the structure of the matured biofilm.

No description available.

The Gram positive soil dwelling bacteria Bacillus subtilis produces spores when encountered with a low nutrient environment. However, B. subtilis can delay spore production by a mechanism known as cannibalism. Cannibalism is a process by which B. subtilis delays commitment to sporulation by killing a subpopulation of its cells. This process involves production of two toxins, SDP and SKF. SDP is a 42 amino acid peptide with a disulfide bond derived from the internal cleavage of its precursor protein pro-SdpC. pro-SdpC is part of the sdpABC operon. Production of extracellular SDP induces expression of the sdpRI operon. Encoded in this operon is the negative regulator SdpR and SdpI. SdpI is a dual function protein which acts both as a signal transduction protein and the immunity factor against SDP. The current model states that production of SDP is sensed via SdpI. SdpI will sequester SdpR to the membrane in response and allow for sdpRI expression. The aims of this dissertation are to establish the requirements for SDP production and its response via SdpI/SdpR during cannibalism. Studies in Chapter II were carried out to determine the factors required for production of the antimicrobial peptide SDP. Site directed mutagenesis of the leader signal peptide sequence in pro-SdpC demonstrated that proper signal peptide cleavage was required for SDP production. Additional site directed mutants of the cysteine residues in pro-SdpC revealed that these are not required for SDP toxic activity. These studies also included deletions within the sdpABC operon and revealed that the two proteins of unknown function, SdpA and SdpB are required for SDP production. Using mass spectrometry analysis, we found that SdpA and SdpB together are required to produce the active 42 amino acid peptide SDP. Taken together we concluded that SDP production was a multi step process which required proteins encoded within the operon and additional processing supplemented in the cell. In Chapter III we investigated the role of SdpI, specifically what residues were required for the signaling and immunity functions observed. Our initial screen, included site directed mutagenesis of highly conserved residues between the 4th and 5th transmembrane domains of SdpI. These resulted in over 20 SdpI mutants generated. From these, only two SdpI mutants had defects in either signal transduction or SDP immunity. Additional localized mutagenesis was used to isolate two other mutants in SdpI which only affected signal transduction or SDP immunity. SdpI signaling-immunity+ mutants presented a defect in SdpR membrane sequestration and sdpRIinduction. Our findings suggest these types of SdpI mutants may be important for the downstream effect of SdpR membrane sequestration. SdpI signaling+ immunity- mutants revealed defects in SDP protection. Some of the residues mutated were conserved in other SdpI homologs. Site directed mutagenesis of these conserved residues in the SdpI ortholog YfhL showed these are also required for SDP resistance. For the first time, we were able to identify mutations which affected only SDP immunity and gained further insight into how SdpI signaling-immunity+ mutants play a role during signal transduction. In Chapter IV we initiated studies to define what regions of the negative regulator SdpR are important for its function during cannibalism. We employed localized mutagenesis to identify SdpR mutants which decreased sdpRIexpression even in the presence of inducing signal. We isolated three such SdpR mutants, referred to as super repressors. We expect these SdpR super repressors are unable to be sequestered to the membrane in the presence of SDP.

Declining antibiotic discovery and flourishing antibiotic resistance have led to a modern antibiotic crisis which threatens to compromise our ability to treat infectious disease. Consequently, there is significant interest in developing new antibiotics with novel modes of action and chemical properties. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural compounds with the appealing attributes of being derived directly from a genetic template while possessing numerous exotic chemical features that contribute to stability and antimicrobial activity. Abundant in nature, their diverse range of biological activities makes them excellent prospects for antibiotic development. Thiopeptides, a RiPP family rich in chemical complexity, represent a particularly promising example. Characterized by post-translationally formed sulfur- and nitrogen-containing heterocycles, more than 100 different thiopeptides have been identified from various cultivable bacterial producers, and the mining of genomic and metagenomic data promises to uncover many more chemical species that have eluded discovery by conventional means. These peptides are potent inhibitors of bacterial protein synthesis and have been shown effective against many drug-resistant pathogens. Despite these attractive properties, therapeutic applications have been limited by the lack of an efficient synthetic route and poor aqueous solubility. Both of these challenges would be greatly alleviated by a more complete understanding of thiopeptide biosynthesis and improved systems for analysis and engineering. Here we describe the characterization of a new thiopeptide gene cluster, which encodes the archetypal thiopeptide micrococcin P1. We describe the identification of the bioactive product and detail the mechanism of immunity in the producing strain. We also describe efforts to engineer this pathway for heterologous expression in Bacillus subtilis. Using this platform, we have been able to dissect this intricate biosynthetic pathway and parse the order and timing of the processing events involved in peptide maturation. The knowledge gained from these studies will inform future efforts to adapt thiopeptides for therapeutic use, and guide efforts to engineer unnatural compounds using the exotic enzymology employed by thiopeptide producing bacteria.

Graduation date: 1994

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Ph.D. / Biochemistry and Molecular Biology / The Bacillus subtilis spx gene encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It resides in the yjbC-spx operon and is transcribed from at least four promoters, three (P[subscript]1, P[subscript]2 and P[subscript]B) residing upstream of yjbC and one (P[subscript]M) located in the intergenic region between yjbC and spx. We uncovered a second intergenic promoter, P[subscript]3, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide, by primer extension analysis. P[subscript]3 is recognized by the σ[superscript]A form of RNA polymerase (RNAP) in vitro without the involvement of a transcriptional activator. Deletion analysis together with point mutation analysis uncovered two negative cis-acting control elements within the P[subscript]3 promoter. Previously published studies and transcription factor/transformation array technology uncovered two transcriptional repressors, PerR and YodB that were potential candidates for the missing trans-acting factors affecting P[subscript]3 promoter utilization. PerR was previously characterized as the regulator of the inducible peroxide stress response in B. subtilis, while YodB is a novel DUF24/MarR type repressor that controls genes that are induced in response to phenolic compounds and oxidative stress. The derepression of spx was detected in both perR and yodB mutants by examining the level of spx expression using the spx-bgaB fusion construct. The additive effect was observed in the perR yodB double mutant. The regions of spx P[subscript]3 DNA required for transcriptional repression by YodB and PerR were confirmed by DNase I footprinting analysis. PerR protects an area from approximately position -3 to +35. YodB binds a region from approximately positions -3 to -32. The binding of YodB and PerR proteins to spx P[subscript]3 promoter DNA was impaired by addition of diamide and H[subscript]2O[subscript]2 in vitro as determined by DNase I footprinting analysis. Besides spx, YodB also controls the divergently transcribed yodC gene which encodes a putative nitroreductase that is induced by disulfide stress. Microarray and proteome analyses were performed to identify other genes controlled by YodB. yocJ (azoR1), encoding the putative FMN-dependent NADH-azoreductase, was the most strongly derepressed by yodB null mutation and was induced in response to diamide, catechol, MHQ and nitrofurantoin stress. bsrB encoding a small 6S RNA located downstream of azoR1, is co-transcribed with azoR1 and increased in concentration in response to thiol-reactive compounds. The yodB mutant confers a catechol and MHQ resistance phenotype due to AzoR1 overproduction. In addition, the yodBmhqR double mutant, bearing the deletion of the mhqR gene encoding a MarR-like repressor, that overproduces AzoR1 and MhqR-regulated paralog AzoR2, exhibits hyper-resistance to thiol-reactive compounds. Thus, the detoxification of thiol-reactive substances in YodB and MhqR regulons show overlapping functions. DNase I footprinting analysis, together with promoter sequence alignments, uncovered YodB boxes which contain a common 15 bp consensus sequence for YodB-DNA interaction. The YodB protein contains three cysteine residues Cys6, Cys101 and Cys108. The conserved Cys6 contributes to the repression of spx and azoR1 transcription by YodB. Moreover, mass spectrometry revealed YodB Cys modifications by catechol and MHQ.

A cloned esterase and several cytoplasmic proteins which lack a classical cleavable signal-peptide were secreted during late stationary phase in B. subtilis. Several lines of evidence indicate that secretion of enolase, SodA, and Est55 is not due to cell lysis. The extent of possible release of these proteins mediated by membrane vesicles into the medium was also found to be minimal. We have identified a hydrophobic α–helical domain within enolase that contributes to the secretion specificity. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid-encoded mutant enolases is totally blocked, while that of the wild-type chromosomal enolase is not affected in the same cultures. However, mutations on the conserved basic residues flanking the hydrophobic core region show no effect. GFP fusion experiments demonstrate that minimal length of N-terminus 140 amino acids and its tertiary structure are required to serve as a functional signal for the export of enolase. Transcriptome analysis revealed several interesting patterns in gene expression when the cell growth switches from exponential phase into stationary phase. As expected, once cell growth enters the stationary phase, expressions of most SigA-dependent house-keeping genes (for syntheses of ATP, amino acids, nucleotides, ribosomes), and surprisingly secY and yidC homolog in the Sec-dependent general protein secretion system were significantly decreased; however, secA and sipT were found progressively induced in the stationary phase. The sigB gene and the SigB regulon exhibited a distinct pattern of transient induction with a peak in transition phase. A total of 62 genes were induced by three fold after cessation of SigB-dependent surge, which includes sigW and many of SigW-depedent genes specifically for antitoxin resistant genes, and some unknown function genes. In addition, oxidative stress response and damage repair genes also dominantly induced in stationary phase implied a high level of oxidant or thio-depleting agents in stationary phase. Besides, induction of fruRAB at T40 and gap operon at T100 suggested a sequential switch of carbon utilization from glucose to fructose. These results indicate a complex adaptation physiology as Bacillus cells change from the fast growing exponential phase toward the stationary phase.

Three experiments were conducted to evaluate the impacts of removing antibiotic growth promoters (AGP) on broiler performance and yield and to evaluate alternative products as potential replacements. In experiment one, approximately 552,000 broilers were reared in four solid-wall, tunnel ventilated houses that were divided into two paired-house facilities, each assigned one of two dietary treatments. The treated group received basal diets containing salinomycin (SAL), roxarsone (ROX) and AGP, while the control group received the same diets without ROX and AGP. Removal of ROX and AGP had no affect on average body weight and feed efficiency, while livability was significantly affected negatively by the removal of ROX and AGP. Tender, wing, drum and percentage of total white meat showed significant improvements in yield during the study, while all other parts were not affected by removal of ROX and AGP. In experiment two, an investigation was conducted to evaluate the effects on performance from feeding Bacillus subtilis spores (Gallipro®, Chr Hansen A/S, Denmark), as a direct-fed microbial additive, to commercial broiler chickens. Birds were divided among two paired-house facilities. The treatment group received basal diets supplemented with B. subtilis spores, while the control group was fed the same basal diets containing an AGP. Feed conversion ratio was significantly lower for the treatment group, while average body weight, coccidiosis lesion scores, and footpad scores were not affected by the treatments. In experiment three, 6,000 broiler chickens were equally divided among four treatment groups and reared to 49 d to determine the effectiveness mannan oligosaccharides (MOS, Bio-Mos®, Alltech, Nicholasville, Kentucky, USA) as an alternative for an AGP program and MOS plus Natustat™ (NAT, Alltech, Nicholasville, Kentucky, USA) as an alternative to an enteric health program (AGP+anticoccidial drug). Average body weight for the control (CON) and antibiotic (ANT) groups was significantly different from the MOS+NAT group, but not the MOS group. Carcass front half, carcass hind half, frame and skin yields were improved for all treatments when compared to the MOS+NAT group. Conversely, percent total white meat yield was improved with the inclusion of MOS when compared to the ANT group. The findings of this research suggest that the removal of AGP from the diets of commercial broiler chickens does not affect the performance and yield of the birds over a one year production period. Furthermore, B. subtilis spores and mannan oligosaccharides provide acceptable alternatives to an AGP program.

Thesis--University of Oklahoma. / Includes bibliographical references (leaves 25-26).