In Escherichia coli, the speC gene encodes biosynthetic ornithine decarboxylase (ODC), an enzyme that catalyzes the decarboxylation of ornithine to produce putrescine. The two polyamines, putrescine and spermidine, and the cyclic AMP (CAMP) - cAMP receptor protein (CRP) are known to inhibit the expression of ODC via undefined mechanisms. A single copy of the speC’-lacZ fusion plasmid pOL-1, containing an 843 base pair fragment including the spec promoter, was transferred to the E. coli CB806 chromosome to create E. coli λCBOL. In cell-free extracts prepared from E. coli λCBOL supplemented with cAMP, putrescine, or spermidine, the B-galactosidase activity encoded by the speC’-lacZ fusion was compared to the ODC activity encoded by spec. Only cyclic AMP and putrescine repressed the speC’-lacZ fusion. Cyclic AMP, putrescine, and spermidine all repressed the spec gene. A 444 bp AluI restriction fragment, containing a putative CRP binding site and a downstream open reading frame (ORF2) present on the strand complementary to speC, was fused to lacZ to create a transcriptional fusion, pCC2L. Analysis of E. coli CB806/pCC2L revealed that there was no detectable β8- galactosidase activity from the ORF2-lacZ fusion. However, promoter activity was detected in the opposite direction (3’ to 5’) of ORF2 as alkaline phosphatase activity, encoded on the same plasmid, increased in the presence of CAMP. A 678 bp DraI-AatII fragment, containing the CRP binding site and an adjacent open reading frame (ORF3) present on the speC coding strand, was subcloned into plasmid pBR322 to create pBCR. In the presence of 10 mM cAMP, E. coli CB806/pBCR exhibited an 18% inhibition in ODC activity encoded by spec. It is proposed that ORF3 encodes a protein that represses speC in the presence of CAMP. / Master of Science

Bacteria have developed specialized responses that involve the expression of particular genes present in a given regulon. Sigma factors provide regulatory mechanisms to respond to stress by acting as transcriptional initiation factors. This work focuses on σ32 during oxidative stress in Escherichia coli. The differential response of key heat shock (HS) genes was investigated during HS and oxidative stress using qPCR techniques. While groEL and dnaJ experienced increases in transcriptional response to H2O2 (10 mM), HS (42°C), and paraquat (50 mM) exposure, the abundance of dnaK over the co-chaperones was apparent. It was hypothesized that DnaK undergoes oxidative modification by reactive carbonyls at its Lys-rich C-terminus, accounting for the differential response during oxidative stress. A σ32-mediated β-galactosidase reporter was devised to detect the activity of wild-type DnaK and DnaKV634X modified to lack the Lys-rich C-terminus. Under unstressed conditions and HS, σ32 was bound at the same rate in both strains. When subjected to H2O2, the WT DnaK strain produced significantly higher β-galactosidase than DnaKV634X (one-tailed Student’s t test p=0.000002, α=0.05) and approached the same level of output as the lacZ positive control. The β-galactosidase assay indicates that DnaK undergoes Lys modification in the WT strain, preventing the protein from binding σ32, increasing the activity of σ32, and resulting in higher β-galactosidase activity than the DnaKV634X strain. In the DnaKV634X strain DnaK continues to bind σ32 so that σ32 could not promote the production of β-galactosidase. These findings demonstrate how DnaK is oxidatively modified, hindering the interaction with σ32 in manner distinct from HS.

Bibliography: leaves 267-287 / xviii, 287, [ca. 40] leaves, [10] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1985

Gene expression heterogeneity underlies critical bacterial phenotypes including antibiotic tolerance, pathogenesis, and communication. Though microbial population heterogeneity has been appreciated for decades, we still lack a complete view of single-cell gene expression and phenotypic states. Various tools, including bulk RNA-seq and proteomics, are available for probing all genes on a population-level. Conversely, fluorescent protein reporters and in situ hybridization can capture single-cell states but only for a limited number of genes. Single-cell RNA-sequencing (scRNA-seq), which can quantify expression of all genes with resolution for individual cells, has revolutionized studies of heterogeneous eukaryotic populations. However, adaptation to bacteria has been hindered by technical barriers. This thesis will describe the development of high-throughput scRNA-seq for bacteria and its application to uncover a distinct transcriptional state of rare antibiotic-tolerant cells called persisters. Chapter 2 presents prokaryotic expression profiling by tagging RNA in situ and sequencing (PETRI-seq), our novel scRNA-seq technology. I will detail how PETRI-seq was optimized to overcome bacteria-specific challenges, including lack of mRNA polyadenylation, thick cell walls, and extremely low mRNA abundance. Using combinatorial indexing, PETRI-seq uniquely barcodes tens of thousands of gram-negative and/or gram-positive cells in a single experiment at low cost. In proof-of-concept experiments, we show robust discrimination of E. coli growth phases and identification of rare prophage activation in S. aureus. PETRI-seq will be broadly useful for characterizing bacterial heterogeneity in many contexts. Chapter 3 describes an expansive investigation into antibiotic persistence in E. coli. When a population is treated with lethal antibiotics, persisters are rare cells that can survive the exposure by assuming a relatively dormant state. Understanding the gene expression state and molecular drivers of persistence has been a longstanding goal with major potential to inform drug development and clinical practice. We have applied PETRI-seq to multiple models of E. coli persistence and discovered a distinct transcriptional state underlying this phenotype. In parallel, we used genome-wide CRISPR-interference to probe the functional contribution of every gene to the persistence phenotype. We discovered multiple driver genes and pathways. Comprehensive validation established Lon protease and YqgE as key gene products modulating translation rate, post-starvation dormancy, and persistence. Our work is a major step in defining the physiological state of persistence and the molecular processes leading cells into this state. In all, this thesis demonstrates how a new generation of systems approaches, including scRNA-seq and CRISPR-interference, enable new discoveries about long-studied phenomena. The overarching approach is broadly applicable with potential to inspire a wide range of microbiology studies.

A functional sense of smell is mediated by Olfactory Receptor proteins (ORs), which reside in olfactory sensory neurons (OSNs) in the epithelium of our nose. Only a singular OR allele out of roughly 2,400 other OR alleles is expressed in every OSN⁽¹˒ ²⁾. Singular expression of the active OR gene occurs in a unique transcriptional hub⁽³⁻⁵⁾. This hub contains one OR promoter and many interchromosomal enhancers that converge upon the hub from far nuclear distances⁽⁵˒ ⁶⁾. Once in the hub, the enhancers work in tandem with each other and with the transcription factors (TFs) Lhx2, EBF, and their cofactor, Ldb1⁽⁵˒ ⁷˒ ⁸⁾ The Greek islands contain a novel “composite” motif containing an Lhx2 and EBF binding site directly next to each other⁽⁸⁾. My work aims to understand how these proteins interact with each other and the composite motif to contribute to the accumulation of many enhancers around only a single promoter in the hub. Our findings illuminate how transcription factor interactions contribute to the hub's unique DNA architecture. To investigate the biochemical foundation of OR hubs, we used 𝑒. 𝑐𝑜𝑙𝑖 to grow and purify full-length and truncated forms of Lhx2, Ebf1, and Ldb1. We used the recombinant proteins with other biochemical methods to characterize the interactions between Lhx2, Ebf1, Ldb1, and different types of DNA found in the OR hub. We used EMSAs to quantify the binding affinity that Lhx2 and Ebf1 have for promoter versus enhancer DNA. Finally, we mixed the purified full-length proteins and used fluorescence microscopy to visualize their behavior in solution. This research combined with in vivo imaging in the Lomvardas lab provides a better understanding as to how molecular interactions 𝑖𝑛 𝑣𝑖𝑡𝑟𝑜 contribute to the hub’s architecture 𝑖𝑛 𝑣𝑖𝑣𝑜, and ultimately, stable OR expression. Our biochemical studies suggest that Lhx2 and Ebf1 can bind at the same time to a single composite motif yet they seem to bind independently of one another. We have used EMSAs to determine that Lhx2 binds much better to OR enhancer DNA compared to Ebf1 and that it might stabilize enhancer contacts. We have also found that Lhx2 and Ebf1 do not cooperatively bind to enhancers- indicating that affinity alone does not explain the accumulation of TFs to the OR hub. Our 𝑖𝑛 𝑣𝑖𝑡𝑟𝑜 imaging shows that Lhx2, Ebf1, and Ldb1 self-assemble into rigid nucleoprotein condensates driven by the composite motif of enhancer DNA. This imaging work also reveals that Lhx2 and Ldb1 are scaffolding proteins with low mobility which drive rigid condensate formation over enhancers. Ebf1 displays more plasticity and turnover into condensates indicating that it plays a more complex role as a recruited factor to these assemblies. We have coupled this data with 𝑖𝑛 𝑣𝑖𝑣𝑜 imaging of endogenous Lhx2, Ebf1, and Ldb1 to find that these factors display similar binding and dynamics 𝑖𝑛 𝑣𝑖𝑣𝑜. This data helps to provide a biophysical model of how OR hubs support multi-enhancer and protein-rich environments that are succinct from their surrounding environment. Our studies suggest that the OR hub forms a rigid phase separated compartment in the nucleus- driven by Lhx2 and Ldb1. This solid-like phase separation may contribute to how singular OR expression is achieved. Such long-range enhancer contacts must stay assembled long-term for continuous OR transcription. Therefore, traditional TF DNA binding dynamics would not explain the longevity of these contacts in the OR hub. This work challenges the traditional model of liquid phase separated nuclear compartments and may provide a broader understanding to how long range and inter-chromosomal compartments are maintained.

Escherichia coli (E. coli) is a common inhabitant of surface waters in the developed and developing worlds. The majority of E. coli cells present in water are not particularly pathogenic to humans; however, there are some present in small proportion that possess virulence genes that allow them to colonize the digestive tract. Pathogenic E. coli causes acute and chronic diarrheal diseases, especially among children in developing countries and in travelers in these locales. The present study, conducted between August 2007 and July 2008, investigated the prevalence and distribution of virulent E. coli strains as either free or attached cells in the final effluents of three wastewater treatment plants located in the Eastern Cape Province of South Africa and its impact on the physico-chemical quality of the receiving water body. The wastewater treatment plants are located in urban (East Bank Reclamation Works, East London), peri-urban (Dimbaza Sewage Treatment Works) and in rural area (Alice Sewage Treatment Works). The effluent quality of the treatment plants were acceptable with respect to pH (6.9-7.8), temperature (13.8-22.0 °C), dissolved oxygen (DO) (4.9-7.8 mg/L), salinity (0.12-0.17 psu), total dissolved solids (TDS) (119-162 mg/ L) and nitrite concentration (0.1-0.4 mg/l). The other xii physicochemical parameters that did not comply with regulated standards include the following: phosphate (0.1-4.0 mg/L); chemical oxygen demand (COD) (5-211 mg/L); electrical conductivity (EC) (237-325 μS/cm) and Turbidity (7.7-62.7 NTU). Results suggest that eutrophication is intensified in the vicinity of the effluent discharge points, where phosphate and nitrate were found in high concentrations. Presumptive E. coli was isolated from the effluent samples by culture-based methods and confirmed using Polymerase Chain Reaction (PCR) techniques. Antibiogram assay was also carried out using standard in vitro methods on Mueller Hinton agar. The viable counts of presumptive E. coli for the effluent samples associated with 180 μm plankton size ranged between 0 – 4.30 × 101 cfu/ml in Dimbaza, 0 – 3.88 × 101 cfu/ml in Alice and 0 – 8.00 × 101 cfu/ml in East London. In the 60 μm plankton size category E. coli densities ranged between 0 and 4.2 × 101 cfu/ml in Dimbaza, 0 and 2.13 × 101 cfu/ml in Alice and 0 and 8.75 × 101 cfu/ml in East London. Whereas in the 20 μm plankton size category presumptive E. coli density varied from 0 to 5.0 × 101 cfu/ml in Dimbaza, 0 to 3.75 × 101 cfu/ml in Alice and 0 to 9.0 × 101 cfu/ml in East London. The free-living presumptive E. coli density ranged between 0 and 3.13 × 101 cfu/ml in Dimbaza, between 0 and 8.0 × 101 cfu/ml in Alice and between 0 and 9.5 × 101 cfu/ml in East London. Molecular analysis successfully amplified target genes (fliCH7, rfbEO157, ial and aap) which are characteristic of pathogenic E. coli strains. The PCR assays using uidA-specific primer confirmed that a genetic region homologous in size to the E. coli uidA structural gene, including the regulatory region, was present in 3 of the E. coli isolates from Alice, 10 from Dimbaza and 8 from East London. Of the 3 E. coli isolates from Alice, 1 (33.3%) was positive for the fliCH7 genes and 3 was positive for rfbEO157 genes. Out of the 10 isolates from Dimbaza, 4 were xiii positive for fliCH7 genes, 6 were positive for the rfbEO157 genes and 1 was positive for the aap genes; and of the 8 isolates from East London, 1 was positive for fliCH7 genes, 2 were for the rfbEO157 genes, 6 were positive for the ial genes. Antimicrobial susceptibility profile revealed that all of the E. coli strains isolated from the effluent water samples were resistant (R) to linezolid, polymyxin B, penicillin G and sulfamethoxazole. The E. coli isolates from Dimbaza (9/10) and East London (8/8) respectively were resistant to erythromycin. All the isolates were found to be susceptible (S) to amikacin, ceftazidime, ciprofloxacin, colistin sulphate, ceftriaxone, cefotaxime, cefuroxime, ertapenem, gatifloxacin, gentamycin, imidazole, kanamycin, meropenem, moxifloxacin, neomycin, netilmicin, norfloxacin and tobramycin. The findings of this study revealed that the Alice wastewater treatment plant was the most efficient as it produced the final effluent with the least pathogenic E. coli followed by the Dimbaza wastewater treatment plant. In addition, the findings showed that the wastewater treatment plant effluents are a veritable source of pathogenic E. coli in the Eastern Cape Province watershed. We suggest that to maximize public health protection, treated wastewater effluent quality should be diligently monitored pursuant to ensuring high quality of final effluents.

CsrA is a global regulator and the main player of the carbon storage regulator (Csr) network, a well- conserved regulatory network in the bacterial world. CsrA is involved in regulation of many physiological processes, including pathways of central carbon metabolism, biofilm formation, motility and virulence in pathogenic species. CsrA acts at the post-transcriptional level by binding specific sequences on its target mRNAs, leading to mRNA destabilization or stabilization. The majority of studies were analyzing a csrA mutant of E. coli K-12 encoding a truncated form of the CsrA protein, retaining residual activity.<p>This work aims at further characterize the roles of CsrA by deleting the entire csrA gene in a recently isolated strain, the uropathogenic E. coli CFT073 strain. Deletion of csrA leads to a marked growth defect, indicating that this gene, although not essential, is primordial for growth. We performed experimental evolution of csrA deletion mutants. Compensatory mutants totally outcompete the original csrA deletion mutant after six days of culture, indicating that the applied selective pressures are strong. The ÄcsrA and three ÄcsrA evolved mutants were extensively analyzed by combining molecular techniques such as genetics, microscopy and use of fluorescent reporters, and global approaches, including comparative proteomics and whole genome sequencing.<p>Our data indicate that csrA deletion strongly affects central metabolism and energy status, constituting an endogenous metabolic stress that, in turn, induces specific stress responses. This work illustrates the interconnection of multiple regulation networks for responding to specific conditions and demonstrates the flexibility of metabolic network to compensate for genetic perturbations in E. coli.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Accurate replication of the genome is essential for reproduction in all cells. However, even under normal conditions, the replication machinery may face a variety of impediments that can prevent it from completing its task. The mechanism by which cells overcome these hurdles is likely to vary depending upon the nature of the obstacle. Both UV irradiation and inactivation of replicative proteins in DnaB can inhibit the progression of the DNA replication machinery. However, the mechanism by which replication recovers following UV irradiation is different from the mechanism of recovery following the inactivation of the replicative proteins. Previous results show that following UVinduced damage in Escherichia coli, the replication fork is maintained and protected from extensive degradation by RecF, RecO, and RecR until replication can resume. By contrast, replication does not recover following inactivation of the replication protein DnaB, and the nascent DNA is extensively degraded irrespective of whether RecF is present. In this study, we verified DNA replication arrest by monitoring the total DNA accumulation and rate of DNA synthesis following UV-induced DNA damage and inactivation of thermosensitive replication alleles, such as dnaB266. We measured the amount of nascent DNA degradation, allowing us to determine how the newly synthesized strand of DNA is affected following replication fork arrest. Our data indicate that following inactivation of DnaB266, the replication fork is not maintained and is subject to extensive degradation. The degradation that occurs after DnaB266 inactivation is partially reduced in the absence of RecF-O-R, RecJ, and ExoI, suggesting that DNA processing by these enzymes occurs after DnaB arrest. In addition, two-dimensional agarose gel analysis revealed that unique structural intermediates accumulated following inactivation of DnaB266. These observations indicate that the recovery of replication when impeded by DNA lesions, such as those produced by UVirradiation, is maintained and processed through mechanisms that do not resemble the events occurring when replication proteins are inactivated.

No description available.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below