
\newpage
\tablefirsthead{\hline \textbf{Strain}	&	\textbf{Description} &	\textbf{Source}\\ \hline \hline}
\tablehead{\hline \textbf{Strain}	&	\textbf{Description} &	\textbf{Source}\\ \hline \hline}
\tabletail{\hline\multicolumn{3}{r}{\small\sl Continued\ldots}\\}
\tablelasttail{\hline}
\bottomcaption{Strains used in this study. $\dagger$JPA410 was made by cloning a \textit{Mlu}I linker (5'-GACGCGTC-3') into the \textit{Eco}RV site near the beginning of \textit{cheY$_3$}, introducing a stop codon at the site of insertion with a new \textit{Mlu}I restriction site, which is expected to produce a truncated peptide of 26 N-terminal amino acids (named \yiii{}*) \cite{shah2000ftb}. *For future reference, strains made by R.E. Sockett were assigned JPA numbers so they can be located in the freezers (\textit{fliM$_1$}(E12D), \textit{fliM$_1$}(E12Q), \textit{fliM$_1$}(S9F) = JPA1249, JPA1250, JPA1251 respectively). \textit{lacI$^q$} denotes the gene encoding the high expression mutant of the \textit{lacI} gene. Tc$^R$, Kn$^R$, Sm$^R$ denote tetracycline, kanamycin, streptomycin resistance respectively.}
   \label{tab:strains}
   \begin{supertabular} {lp{9cm}c}
XL-1 Blue						& General \ec{} cloning strain. \textit{lacI$^q$}, Tc$^R$																&	Stratagene								\\ \hline
M15pREP4						& \ec{} expression host containing pREP4. Kn$^R$																				&	Qiagen										\\ \hline
S17-1$\lambda$-pir	&	\ec{} strain capable of mobilizing the suicide vector pK18\textit{mobsacB} into \rs{}. Sm$^R$.	& \cite{penfold1992isv} \\ \hline
WS8N								&	Spontaneous nalidixic acid resistant mutant of wild-type WS8													&	\cite{sockett1991ica}			\\ \hline 
JPA410							&	WS8N derivative that carries a \textit{cheY$_3$} gene that has been insertionally inactivated (named \textit{cheY$_3$*})$\dagger$	&	\cite{shah2000ftb}	\\ \hline
JPA421							&	$\Delta$\textit{cheY$_4$} derivative of WS8N																					&	\cite{shah2000ftb}				\\ \hline 
JPA425							&	\textit{cheY$_3$*}, $\Delta$\textit{cheY$_4$} derivative of WS8N											&	\cite{shah2000ftb}				\\ \hline 
JPA1025							&	$\Delta$\textit{cheY}$_1$ $\Delta$\textit{cheY}$_2$ $\Delta$\textit{cheY}$_3$ $\Delta$\textit{cheY}$_4$ $\Delta$\textit{cheY}$_5$ derivative of WS8N	& \cite{porter2006crs}			\\ \hline
JPA1337							&	$\Delta$\textit{cheY}$_1$ $\Delta$\textit{cheY}$_2$ $\Delta$\textit{cheY}$_3$ $\Delta$\textit{cheY}$_4$ $\Delta$\textit{cheY}$_5$ $\Delta$\textit{cheY}$_6$ derivative of WS8N	& \cite{porter2006crs}			\\ \hline
JPA443							&	$\Delta$\textit{che}Op$_1$ and $\Delta$\textit{cheY}$_5$ derivative of WS8N						&	S.L. Porter								\\ \hline 
JPA1349							&	$\Delta$\textit{che}BRA, $\Delta$RSP2230 derivative of WS8N									&	S.L. Porter								\\ \hline
JPA1278							&	$\Delta$\textit{fliM$_2$} (RSP6099) derivative of WS8N																&	S.L. Porter								\\ \hline
\textit{fliM$_1$}(E12D)				&	\textit{fliM$_1$}(E12D) derivative of WS8N*																						&	R.E. Sockett		\\ \hline
\textit{fliM$_1$}(E12Q)				&	\textit{fliM$_1$}(E12Q) derivative of WS8N*																						&	R.E. Sockett		\\ \hline
\textit{fliM$_1$}(S9F)				&	\textit{fliM$_1$}(S9F) derivative of WS8N*																						&	R.E. Sockett		\\ \hline
JPA419							&	$\Delta$\textit{che}Op$_2$ and $\Delta$\textit{cheY}$_4$ derivative of WS8N						&	\cite{shah2000ftb}				\\ \hline 
JPA1301							&	$\Delta$\textit{che}Op$_3$ derivative of WS8N																					&	\cite{porter2002tcl}			\\ \hline
JPA1336							&	$\Delta$\textit{cheY$_6$} derivative of WS8N																					&	\cite{porter2002tcl}			\\ \hline
JPA1314							&	$\Delta$\textit{cheA$_3$} derivative of WS8N																					&	\cite{porter2002tcl}			\\ \hline
JPA1353							&	$\Delta$\cheopi{} (\textit{che}Y$_5$, \textit{che}D, \textit{che}P$_1$, \textit{che}Y$_1$, \textit{che}A$_1$, \textit{che}W$_1$, \textit{che}R$_1$, \textit{che}Y$_2$), $\Delta$\cheopii{} (\textit{che}P$_2$, \textit{che}Y$_3$, \textit{che}A$_2$, \textit{che}W$_2$, \textit{che}W$_3$, \textit{che}R$_2, $\textit{che}B$_1$, \textit{tlp}C), $\Delta$\cheopiii{} (\textit{che}A$_4$, \textit{che}R$_3$, \textit{che}B$_2$, \textit{che}W$_4$, \textit{ppf}A, \textit{tlp}T, \textit{che}Y$_6$, \textit{che}A$_3$), $\Delta$\textit{che}BRA, $\Delta$\textit{che}Y$_4$	&	S.L. Porter								\\ \hline
JPA1319							& $\Delta$\textit{cheA$_2$}, $\Delta$\textit{cheA$_3$} derivative of WS8N								&	S.L. Porter								\\ \hline
JPA1213							&	\textit{cheY$_6$(D56N)} derivative of WS8N																							& \cite{porter2006crs}			\\ \hline
JPA1216							&	\textit{cheY$_6$(D56A)} derivative of WS8N																							& \cite{porter2006crs}			\\ \hline
JPA1218							&	\textit{cheY$_3$(D53N)} derivative of WS8N																							& \cite{porter2006crs}			\\ \hline
JPA1220							&	\textit{cheY$_4$(D53N)} derivative of WS8N																							& \cite{porter2006crs}			\\ \hline
JPA917							&	\textit{cheY$_3$*}, \textit{cheY$_4$}(D53A) derivative of WS8N (parental = JPA425)											&	This study								\\ \hline
JPA918							&	\textit{cheY$_3$*}, \textit{cheY$_4$}(D53N) derivative of WS8N (parental = JPA425)										&	This study								\\ \hline
JPA919							&	\textit{cheY$_3$}(D53A), \textit{cheY$_4$}(D53A) derivative of WS8N (parental = JPA917)								&	This study								\\ \hline
JPA920							&	\textit{cheY$_3$}(D53N), \textit{cheY$_4$}(D53N) derivative of WS8N	(parental = JPA918)									&	This study								\\ \hline
\end{supertabular}

\newpage
\tablefirsthead{\hline \textbf{Plasmid}	&	\textbf{Description} &	\textbf{Source}\\ \hline \hline}
\tablehead{\hline \textbf{Plasmid}	&	\textbf{Description} &	\textbf{Source}\\ \hline \hline}
\tabletail{\hline\multicolumn{3}{r}{\small\sl Continued\ldots}\\}
\tablelasttail{\hline}
\bottomcaption{Plasmids used in this study. Kn$^R$, Ap$^R$ denote ampicillin and kanamycin resistance respectively. P$_{A1/04/03}$ denotes an isopropyl ß-D-1-thiogalactopyranoside (IPTG) inducible promoter. $\dagger$See Appendix ~\ref{sec:Plasmid Map - pIND4}, Page ~\pageref{sec:Plasmid Map - pIND4} for pIND4 plasmid map.}
   \par
   \label{tab:plasmids}
   \begin{supertabular}{p{3cm} p{8.5cm} c}
pUC19										&	High copy number cloning vector. Ap$^R$																						&	Pharmacia							\\ \hline
pREP4										&	Plasmid carrying the \textit{lacI} gene.																			&	Qiagen \\ \hline
pIND4										&	\rs{} inducible expression plasmid$\dagger$, P$_{A1/04/03}$, \textit{lacI$^q$}, Kn$^R$		&	\cite{ind2009}				\\ \hline
pK18\textit{mobsacB}		&	Allelic exchange suicide vector. Kn$^R$, Sucrose sensitive								&	\cite{schafer1994smm}	\\ \hline
pK18Y3D53A							&	Construct for replacing \textit{cheY$_3$} with \textit{cheY$_3$}(D53A) in \rs{} genome; pK18\textit{mobsacB} derivative		&	\cite{porter2006crs}		\\ \hline
pK18Y3D53N							&	Construct for replacing \textit{cheY$_3$} with \textit{cheY$_3$}(D53N) in \rs{} genome; pK18\textit{mobsacB} derivative		&	\cite{porter2006crs}		\\ \hline
pK18Y4D53A							&	Construct for replacing \textit{cheY$_4$} with \textit{cheY$_4$}(D53A) in \rs{} genome; pK18\textit{mobsacB} derivative		&	\cite{porter2006crs}		\\ \hline
pK18Y4D53N							&	Construct for replacing \textit{cheY$_4$} with \textit{cheY$_4$}(D53N) in \rs{} genome; pK18\textit{mobsacB} derivative		&	\cite{porter2006crs}		\\ \hline
pQEY1										& \yi{} expression plasmid. pQE30 derivative 																					&	\cite{shah2000ifc}	\\ \hline
pQEY2										& \yii{} expression plasmid. pQE30 derivative 																				&	\cite{shah2000ifc}	\\ \hline
pQEY3										& \yiii{} expression plasmid. pQE30 derivative 																				&	\cite{shah2000ifc}	\\ \hline
pQEY4										& \yiv{} expression plasmid. pQE30 derivative 																				&	\cite{shah2000ifc}	\\ \hline
pQEY5										& \yv{} expression plasmid. pQE30 derivative 																					&	\cite{porter2004crs}	\\ \hline
pQEY6 									&	\yvi{} expression plasmid. pQE30 derivative 																					&	\cite{porter2002tcl}	\\ \hline
pQEY6D56N		 						&	\yvi{}(D56N) expression plasmid. pQE30 derivative																		& \cite{porter2006crs}	\\ \hline
pQEYFP-Y6 							&	YFP-\yvi{} expression plasmid. pQE30 derivative																			& \cite{porter2006crs}	\\ \hline
pIND-empty							&	As pIND4 above, i.e. empty vector used as a control																	&	\cite{ind2009}				\\ \hline
pIND-Y1									&	\textit{cheY$_1$} cloned into pIND4																									&	This study						\\ \hline
pIND-Y2									&	\textit{cheY$_2$} cloned into pIND4																									&	This study						\\ \hline
pIND-Y3									&	\textit{cheY$_3$} cloned into pIND4																									&	This study						\\ \hline
pIND-Y3D53A							&	\textit{cheY$_3$}(D53A) cloned into pIND4																						&	This study						\\ \hline
pIND-Y3D53N							&	\textit{cheY$_3$}(D53N) cloned into pIND4																						&	This study						\\ \hline
pIND-Y4									&	\textit{cheY$_4$} cloned into pIND4																									&	This study						\\ \hline
pIND-Y4D53A							&	\textit{cheY$_4$}(D53A) cloned into pIND4																						&	This study						\\ \hline
pIND-Y4D53N							&	\textit{cheY$_4$}(D53N) cloned into pIND4																						&	This study						\\ \hline
pIND-Y5									&	\textit{cheY$_5$} cloned into pIND4																									&	This study						\\ \hline
pIND-Y6									&	\textit{cheY$_6$} cloned into pIND4																									&	This study						\\ \hline
pIND-Y6D56A							&	\textit{cheY$_6$}(D56A) cloned into pIND4																						&	This study						\\ \hline
pIND-Y6D56N							&	\textit{cheY$_6$}(D56N) cloned into pIND4																						&	This study						\\ \hline
pIND-YFPY6							&	\textit{yfp-cheY$_6$} cloned into pIND4																							&	This study						\\ \hline
pIND-YFPY6D56A					&	\textit{yfp-cheY$_6$}(D56A) cloned into pIND4																				&	This study						\\ \hline
pIND-ecY								&	\ec{} \textit{cheY} cloned into pIND4																									&	This study						\\ \hline
pBAD-ecYD13K						& \ec{} \textit{cheY}(D13K) expression plasmid. pBAD24 derivative											&	Y. Sowa								\\ \hline
pIND-ecY**							&	\ec{} \textit{cheY}(D13K, Y106W) cloned into pIND4																		&	This study						\\ \hline
pIND-ecYZ								&	\ec{} \textit{cheY, cheZ} cloned into pIND4																						&	This study						\\ \hline
pIND-ecZ								&	\ec{} \textit{cheZ} cloned into pIND4																									&	This study						\\ \hline
\end{supertabular}

\Subsubsection{Standard DNA Techniques}
\textit{Preparation of Plasmid DNA} - Carried out using the QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer's instructions.
\newline \newline
\noindent \textit{Restriction Digests} - Carried out using enzymes from New England Biolabs (NEB); 0.5 $\mu$l of each enzyme, 2 $\mu$l 10X buffer, 2-4 $\mu$l DNA to be digested, made up with sterile water to a total volume of 20 $\mu$l.
\newline \newline
\textit{Gel Electrophoresis} - Multi purpose agarose (Roche) was made up at between 0.8 and 2 \% in 0.5X TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.0) and electrophoresed at between 100 and 150 V. The gels were stained for 20 mins in 1 ng/ml ethidium bromide and visualised on a UV transilluminator.
\newline \newline
\textit{Purification of DNA Fragments} - DNA fragments were cleaned from agarose gels or from solution using the Genelute Gel Extraction Kit (Sigma) according to the manufacturer's instructions.
\newline \newline
\textit{Ligations} - 2 $\mu$l T4 DNA ligase (NEB) was used in a final volume of 50 $\mu$l with vector and insert DNA in a ratio of about 1:3 and the supplied reaction buffer and incubated at 15 \degc{} for at least 16 hours.
\newline \newline
\textit{Competent \ec{} Cells} - Prepared using the calcium chloride method of Sambrook and Russell \cite{sambrook2001mcl} and stored in 200 $\mu$l alliquots at -80 \degc{}.
\newline \newline
\textit{Transformation} - DNA was transformed by incubation with 200 $\mu$l of the competent cells on ice for 1 hour, followed by 2 min at 42 \degc{} and 2 min on ice. 1 ml of LB was added and incubated at 37 \degc{} for 1 hour. The mixture was spread on LB-agar plates containing appropriate antibiotics.
\newline \newline
\textit{Polymerase Chain Reaction (PCR)} - Reactions consisted of DNA template ($\sim$100 ng), 100 pmol of each primer (Sigma-Genosys), 12.5 nmol dNTPs, and 1 unit of \textit{pfu} DNA polymerase (Promega), made up to a final volume of 25 $\mu$l. Thermocycling was performed in a Thermal MiniCycler (MJ Research) as follows:
\begin{table}[htbp]
\begin{tabular}{ll}
   Step 1 &         98\degc{} x 5 min \\
 Step 2.1 &         98\degc{} x 2 min \\
 Step 2.2 &         55\degc{} x 1.5 min \\
 Step 2.3 &         72\degc{} x 4 min \\
 Step 3 & repeat step 2, 25 times           \\
 Step 4 &         72\degc{} x 5 min\\
\end{tabular}  
		\label{tab:PCR}
\end{table}

\noindent \textit{DNA Sequencing} - All constructs were sequenced before use by the Automated DNA Sequencing Service (Geneservice, Source BioScience) using BigDye$^{TM}$ dye terminators (PE Biosystems) on a 3730 DNA sequencer (Applied Biosystems). Sequences were assembled using the Staden Software Package and analysed using Clone Manager Software (Scientific and Educational Software).
\newline \newline
\textit{DIG Labelling of DNA Probes for Southern Blot} - DNA for use as a probe in Southern blots was purified from agarose gels as described previously and labelled using the random hexanucleotide priming technique with the DIG DNA Labelling and Detection Kit (Roche) according to the manufacturer's instructions.
\newline \newline
\textit{Southern Blotting} - DNA for Southern blotting was separated on an agarose gel, depurinated, denatured, neutralised and transferred to Hybond-N membrane (Amersham) by capillary transfer as described in \cite{sambrook2001mcl}. The membrane was dried, UV fixed, prehybridised for at least 3 hours. Hybridisation of the probe was carried out at 68 \degc{} in a Hybaid oven over night. Detection was performed using the DIG colour detection kit (Roche) according to the manufacturer's instructions.
\newline \newline
\textit{Conjugation} - Plasmids were conjugated into \rs{} from the donor strain \ec{} S17-1$\lambda$-pir. A stationary phase culture of S17-1 containing the appropriate plasmid was diluted 1:30 into fresh medium and grown at 37 \degc{} to an optical density (OD) of OD$_{600}$ $\sim$0.1. 1 ml of the \ec{} culture and 1 ml of stationary phase \rs{} were centrifuged at 4000 g, washed gently in succinate medium and finally resuspended in 100 $\mu$l of succinate medium. 10 $\mu$l of \ec{} was mixed with 100 $\mu$l of \rs{} cells and pipetted onto a 0.45 $\mu$m filter (Whatman) on an LB-agar plate. After overnight incubation at 30 \degc{} the filter was washed with 1 ml of succinate medium and plated on 1.5 \% LB-agar plates containing kanamycin.
\newline \newline
\textit{\rs{} Chromosomal DNA Preparation} - 1.5 ml of cells were spun down and frozen in liquid nitrogen. The pellet was resuspended in 0.5 ml of 65 \degc{} lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 \% SDS) and 0.1 mg proteinase K added. This was incubated at 42 \degc{} for 2 hours and extracted twice with 0.5 ml phenol:chloroform:isoamyl alcohol (25:24:1 v/v). The DNA was precipitated with 100 \% ethanol, washed in 70 \% ethanol and dried. The pellet was resuspended in 30 $\mu$l of water with RNAase and stored at 4 \degc{} until required.
