Phillip Lee has defected from the Conservatives to the Liberal Democrats, crossing the floor in the House of Commons as Boris Johnson addressed MPs.
The defection of the MP for Bracknell means the government no longer has a working majority.
Lee is the fourth MP to defect to Jo Swinson’s party in the last few months and takes their total to 15.
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Speaking in the Commons, Johnson told Lee he wished him “all the best” after watching him sit down with the Lib Dems.
In a statement, the pro-second referendum former Tory minister accused Johnson of “feeding division and populism” and of using “political manipulation, bullying and lies”.
“This Conservative government is aggressively pursuing a damaging Brexit in unprincipled ways,” he said.
“It is putting lives and livelihoods at risk unnecessarily and it is wantonly endangering the integrity of the United Kingdom.”
It comes ahead of the showdown vote in the Commons on Tuesday evening when opposition at Tory rebels will attempt to seize control of the parliamentary agenda to block a no-deal Brexit on October 31.
Johnson has dramatically raised the stakes by signalling he will ask for a snap general election if the government is defeated.
In a sign of the bitter divisions within the Conservative ranks, former chancellor Philip Hammond accused Downing Street of “rank hypocrisy” and warned of the “fight of a lifetime” if officials attempt to prevent him from standing at the next general election as a Conservative candidate.
Dominic Grieve, who served as attorney general in David Cameron’s government, said threats to withdraw the whip from any Tories voting against the government demonstrated Johnson’s “ruthlessness” in power.
Ex-education secretary Justine Greening and former Foreign Office minister Alistair Burt have both said they will quit at the next election.