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– Myxococcus xanthus is an important soil bacteria, preying on other bacteria using hydrolytic enzymes in swarms of cells known as biofilms

– Their complex life cycle involving biofilm formation and sporulation makes their gene expression mechanisms an interesting topic of study (Fig. 1)

– The M. xanthus genes MXAN_0179, MXAN_0178, and MXAN_0177 are located adjacent to each other with MXAN_0180 as an enhancer binding protein (EBP) gene (Fig. 1)• MXAN_0179 codes for a metallo-beta-lactamase and

has a σ54 promoter, which thus requires an EBP for transcription

• Shown to be down-regulated during sporulation

IntroductionDepartment of Biology, The College of New Jersey, Ewing, NJ 08618

Brian Gaffney and Zaara Sarwar

Characterization of the role of MXAN_0179 metallo-beta lactamase in biofilm formation of Myxococcus xanthus

Identification of transcription regulators of MXAN_0179

– No previous work has been done on these genes thatconfirmed or denied their status as an operon or showed the EBP responsible for MXAN_0179 transcription

– It has been shown that MXAN_0179 can be downregulated while MXAN_0178 and MXAN_0177 are upregulated

– MXAN_0180 is in very close proximity to MXAN_0179– I hypothesize that the EBP MXAN_0180 is involved in the

transcription of MXAN_0179 through its σ54 promoter, but not for the transcription of MXAN_0178 and MXAN_0177 as they do not form an operon (Fig. 2)

Characterizing function of MXAN_0179 - MXAN_0177

– To test if this cluster of genes forms an operon, I plan to use reverse-transcriptase PCR (RT-PCR)

– Primers will be designed to attach intergenically to the cluster ofgenes

– This allows for the detection of cotranscription by showing PCR products only if the genes are cotranscribed, which happens in operons (Fig. 5)

– In addition, the function of these genes can be characterized– Using a beta-lactam antibiotic like penicillin, M. xanthus cells will be

exposed to the antibiotic during different life stages– Expression of the genes will be detected by quantitative RT-PCR (qRT-

PCR)– Beta—lactams can trigger the release of muropeptides into the

periplasm, leading to cell wall recycling and beta-lactamase induction through the three-protein pathway AmpG-AmpR-AmpC

– Because of the connection between cell wall recycling and induction of beta-lactamases, I suspect that expression will be low during dormancy stages while it will be high in developmental and growth stages such as predatory biofilm formation.

– To determine the EBP responsible for MXAN_0179 transcription, I plan to use electrophoretic mobility shift assays (EMSA’s)

– This allows for the detection of promoter binding by showing change in migration distance on a gel

– Purified MXAN_0180 will be run a gel with either the MXAN_0179 promoter region or a nonspecific promoter region as a control (Fig. 3)

ReferencesBenson, G. 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids

Research 27:573–580.Bush, M., and R. Dixon. 2012. The Role of Bacterial Enhancer Binding Proteins as Specialized

Activators of σ54-Dependent Transcription. Microbiology and Molecular Biology Reviews 76:497–529.

Kanehisa, M., and S. Goto. 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Research 28:27–30.

O’Connor, K. A., and D. R. Zusman. 1991. Development in Myxococcus xanthus involves differentiation into two cell types, peripheral rods and spores. Journal of Bacteriology 173:3318–3333.

Palzkill, T. 2013. Metallo-β-lactamase structure and function. Annals of the New York Academy of Sciences 1277:91–104.

Sarwar, Z., B. R. Lundgren, M. T. Grassa, M. X. Wang, M. Gribble, J. F. Moffat, and C. T. Nomura. 2016. GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1. mSphere 1:e00020-16.

Sheng, D., Y. Wang, Z. Jiang, D. Liu, and Y. Li. 2020. ImuA participates in SOS mutagenesis by interacting with RecA1 and ImuB in Myxococcus xanthus. bioRxiv.

Zeng, X., and J. Lin. 2013. Beta-lactamase induction and cell wall metabolism in Gram-negative bacteria. Frontiers in microbiology 4:128.

Figure 1. Life cycle of M. xanthus. M. xanthus cells prey upon other bacteria until no food is left, then aggregate after about 6-18 hours and finally form spores. Once nutrients are available again, the spores germinate and begin the cycle again.

ZOOMhttps://tcnj.zoom.us/j/99375983939?pwd=NHRaL1ZCN3k5dStCNzdJc0lnbW5ydz09

Figure 3. A. schematic of protein expression and purification. B. Proposed result of EMSA should MXAN_0180 be the correct EBP for MXAN_0179. Upward shift in the presence of MXAN_0179 promoter but not in the nonspecific probe indicates specific binding to the MXAN_0179 promoter. – Once the EBP responsible for the transcription of MXAN_0179 is

identified, I plan to test its nucleotide sequence recognition – EBP’s are known to bind to tandem repeats in the promoter of

the gene they regulate– 189 nucleotides upstream of the MXAN_0179 gene lie 3

consecutive tandem repeats that I suspect to be the binding site (Fig. 4)

– By introducing single substitution mutations in these positions and then testing the binding using EMSA’s, I will determine the relative importance of each nucleotide to further characterize the binding of the EBP

Figure 4. Consensus sequence of the proposed MXAN_0180 binding site derived from the three 11-bp tandem repeats in the PMXAN_0179 sequence constructed with WebLogo 3.7.4.

Figure 5. Schematic of RT-PCR methodology of operon testing. MXAN_0179, 0178, and 0177 are either cotranscribed into RNA (left) or separately transcribed (right). If they are cotranscribed (i.e.form an operon), intergenic cDNA products A and B can be formed.

Figure 2. Diagram of the transcription of MXAN_0179 with its σ54 promoter and MXAN_0180 as the proposed EBP. The EBP loops the DNA (leading to binding in the opposite direction) to bring itself in proximity with the σ54 factor, where it couples the energy from ATP hydrolysis to the isomerization of σ54-RNAP closed complex to open complex configuration.

A B